Strains used in whole organism Plasmodium falciparum vaccine trials differ in genome structure, sequence, and immunogenic potential

Abstract

Background: Plasmodium falciparum (Pf) whole-organism sporozoite vaccines have been shown to provide significant protection against controlled human malaria infection (CHMI) in clinical trials. Initial CHMI studies showed significantly higher durable protection against homologous than heterologous strains, suggesting the presence of strain-specific vaccine-induced protection. However, interpretation of these results and understanding of their relevance to vaccine efficacy have been hampered by the lack of knowledge on genetic differences between vaccine and CHMI strains, and how these strains are related to parasites in malaria endemic regions.

Methods: Whole genome sequencing using long-read (Pacific Biosciences) and short-read (Illumina) sequencing platforms was conducted to generate de novo genome assemblies for the vaccine strain, NF54, and for strains used in heterologous CHMI (7G8 from Brazil, NF166.C8 from Guinea, and NF135.C10 from Cambodia). The assemblies were used to characterize sequences in each strain relative to the reference 3D7 (a clone of NF54) genome. Strains were compared to each other and to a collection of clinical isolates (sequenced as part of this study or from public repositories) from South America, sub-Saharan Africa, and Southeast Asia.

Results: While few variants were detected between 3D7 and NF54, we identified tens of thousands of variants between NF54 and the three heterologous strains. These variants include SNPs, indels, and small structural variants that fall in regulatory and immunologically important regions, including transcription factors (such as PfAP2-L and PfAP2-G) and pre-erythrocytic antigens that may be key for sporozoite vaccine-induced protection. Additionally, these variants directly contributed to diversity in immunologically important regions of the genomes as detected through in silico CD8⁺ T cell epitope predictions. Of all heterologous strains, NF135.C10 had the highest number of unique predicted epitope sequences when compared to NF54. Comparison to global clinical isolates revealed that these four strains are representative of their geographic origin despite long-term culture adaptation; of note, NF135.C10 is from an admixed population, and not part of recently formed subpopulations resistant to artemisinin-based therapies present in the Greater Mekong Sub-region.

Conclusions: These results will assist in the interpretation of vaccine efficacy of whole-organism vaccines against homologous and heterologous CHMI.

Keywords: Genome assembly; Malaria; P. falciparum; PfSPZ vaccine; Whole-sporozoite vaccine.

Fig. 1

PacBio Assemblies for each PfSPZ strain reconstruct entire chromosomes in one to three continuous pieces. To determine the likely position of each non-reference contig on the 3D7 reference genome, MUMmer’s show-tiling program was used with relaxed settings (-g 100000 -v 50 -i 50) to align contigs to 3D7 chromosomes (top). 3D7 nuclear chromosomes [–14] are shown in gray, arranged from smallest to largest, along with organelle genomes (M = mitochondrion, A = apicoplast). Contigs from each PfSPZ assembly (NF54: black, 7G8: green, NF166.C8: orange, NF135.C10: hot pink) are shown aligned to their best 3D7 match. A small number of contigs could not be unambiguously mapped to the 3D7 reference genome (unmapped)

Fig. 2

Distribution of polymorphisms in PfSPZ PacBio assemblies. Single nucleotide polymorphism (SNP) densities (log SNPs/ 10 kb) are shown for each assembly; the scale [0–3] refers to the range of the log-scaled SNP density graphs—from 10⁰ to 10³. Inner tracks, from outside to inside, are NF54 (black), 7G8 (green), NF166.C8 (orange), and NF135.C10 (pink). The outermost tracks are the 3D7 reference genome nuclear chromosomes (chrm1 to chrm 14, in blue), followed by 3D7 genes on the forward and reverse strand (black tick marks). Peaks in SNP densities mostly correlate with subtelomeric regions and internal multi-gene family clusters

Fig. 3

Comparison of predicted CD8⁺ T cell epitopes from pre-erythrocytic antigen amino acid sequences. CD8⁺ T cell epitopes were predicted in silico for 42 confirmed or suspected pre-erythrocytic antigens (See Additional file 2: Table S7 for a complete list of genes included in this analysis). The plot shows the number of shared or unique epitopes, as compared between different PfSPZ strain groupings. The height of the bar is the number of epitopes that fell into each intersection category, and the horizontal tracks below the bars show the PfSPZ strains that are included in that intersection. For example, the first bar represents the number of shared epitopes between NF54, 7G8, and NF135.C10. At the bottom left, colored tracks represent the total number of epitopes predicted across all genes (> 10 k for each strain). As the vast majority of predicted epitopes were shared among all four strains, that group was removed from the bar plot to achieve better visual definition for the other comparison

Fig. 4

Predicted CD8⁺ T cell epitopes in the P. falciparum circumsporozoite protein (PfCSP). Protein domain information based on the 3D7 reference sequence of PfCSP is found in the first track. The second track are previously experimentally validated (Exp. Val.) epitopes (from [59], after removing duplicate epitope sequences and epitopes > 20 amino acids in length) and the following tracks are epitopes predicted in the PfCSP sequences of NF54, 7G8, NF166.C8, and NF135.C10, respectively. Each box is a sequence that was identified as an epitope, and colors represent the HLA type that identified the epitope. The experimentally validated epitopes do not have HLA types reflected and are simply jittered across two rows

Fig. 5

Global diversity of clinical isolates and PfSPZ strains. Principal coordinate analyses (PCoA) of clinical isolates (n = 654) from malaria-endemic regions and PfSPZ strains were conducted using biallelic non-synonymous SNPs across the entire genome (left, n = 31,761) and in a panel of 42 pre-erythrocytic genes of interest (right, n = 1060). For the genome-wide dataset, coordinate 1 separated South American and African isolates from Southeast Asian and Papua New Guinean isolates (27.6% of variation explained), coordinate two separated African isolates from South American isolates (10.7%), and coordinate three separated Southeast Asian isolates from Papua New Guinea (PNG) isolates (3.0%). Similar trends were found for the first two coordinates seen for the pre-erythrocytic gene data set (27.1 and 12.6%, respectively), but coordinate three separated isolates from all three regions (3.8%). In both datasets, NF54 (black cross) and NF166.C8 (orange cross) cluster with West African isolates (isolates labeled in red and dark orange colors), 7G8 (bright green cross) cluster with isolates from South America (greens and browns), and NF135.C10 (pink cross) clusters with isolates from Southeast Asia (purples and blues)

Fig. 6

NF135.C10 is part of an admixed population of clinical isolates from Southeast Asia. Top: admixture plots for clinical isolates from Myanmar (n = 16), Thailand (n = 34), Cambodia (n = 109), Papua New Guinea (PNG, n = 34), and NF135.C10 (represented by a star) are shown. Each sample is a column, and the height of the different colors in each column corresponds to the proportion of the genome assigned to each K population by the model. Bottom: hierarchical clustering of the Southeast Asian isolates used in the admixture analysis (branch and leaves colored by their assigned subpopulation) and previously characterized Cambodian isolates (n = 167, black; [64]) place NF135.C10 (star) with samples from the previously identified KHA admixed population (shown in gray dashed box). The y-axis represents distance between clusters