Background A highly effective vaccine for malaria remains an elusive target, at least in part due to the under-appreciated natural parasite variation. This study aimed to investigate genetic and structural variation, and immune selection of leading malaria vaccine candidates across the Plasmodium falciparum’s life cycle.

Methods We analyzed 325 P. falciparum whole genome sequences from Zambia, in addition to 791 genomes from five other African countries available in the MalariaGEN Pf3k Rdatabase. Ten vaccine antigens spanning three life-history stages were examined for genetic and structural variations, using population genetics measures, haplotype network analysis, and 3D structure selection analysis.

Findings Among the ten antigens analyzed, only three in the transmission-blocking vaccine category display P. falciparum 3D7 as the dominant haplotype. The antigens AMA1, CSP, MSP119 and CelTOS, are much more diverse than the other antigens, and their epitope regions are under moderate to strong balancing selection. In contrast, Rh5, a blood stage antigen, displays low diversity yet slightly stronger immune selection in the merozoite-blocking epitope region. Except for CelTOS, the transmission-blocking antigens Pfs25, Pfs48/45, Pfs230, Pfs47, and Pfs28 exhibit minimal diversity and no immune selection in epitopes that induce strain-transcending antibodies, suggesting potential effectiveness of 3D7-based vaccines in blocking transmission.

Interpretations These findings offer valuable insights into the selection of optimal vaccine candidates against P. falciparum. Based on our results, we recommend prioritizing conserved merozoite antigens and transmission-blocking antigens. Combining these antigens in multi-stage approaches may be particularly promising for malaria vaccine development initiatives.

Funding Purdue Department of Biological Sciences; Puskas Memorial Fellowship; National Institute of Allergy and Infectious Diseases (U19AI089680).

Evidence before this study Decades of research on the most virulent malaria parasite, Plasmodium falciparum, have yielded multiple antigen candidates of pre-erythrocytic, blood-stage, and transmission-blocking vaccines in varying stages of development from preclinical development to more advanced clinical trials. The malaria vaccine, RTS,S/AS01, which was constructed using the C-terminal and NANP repeat region of the Circumsporozoite Protein (CSP) from the African reference strain 3D7, was approved and recommended for use in 2021. However, the vaccine’s lower efficacy is likely a result of the genetic polymorphism of the target antigen shown by studies on natural variation in CSP. Similarly, another more recent pre-erythrocytic vaccine, R21/Matrix-M, showed great promise in clinical trials and was recommended in late 2023 by the WHO for use for prevention of malaria in children, but is also multi-dose and CSP-based. To maximize vaccine efficacy, it would be more strategic to first understand diversity and variation of antigens across the three types of vaccine classes, targeting various stages of the P. falciparum life cycle. Previous studies have reported analyses of vaccine candidate antigens but were mostly limited to pre-erythrocytic and blood-stage antigens, with less focus on transmission-blocking antigens. These studies revealed that most of the pre-erythrocytic and blood-stage antigens are of high diversity due to balancing selection, posing challenges for vaccine design to encompass the antigenic variation.

A search conducted on PubMed on April 1, 2024, for relevant published research which used the terms “malaria vaccine”, “Plasmodium falciparum” [not “vivax”], “selection” and “diversity” yielded 48 studies between 1996 and the present day, with only 14 published studies in the past 3 years. This emphasizes the need for more studies assessing genetic diversity and selection of potential P. falciparum vaccine candidates to aid in more effective vaccine development efforts. A similar search with the terms “transmission-blocking vaccine”, “malaria”, “Plasmodium falciparum”, not “vivax”, “selection” and “diversity” without any date or language restrictions revealed three relevant studies. This warrants future studies to explore transmission-blocking vaccines in this context.

Added value of this study By comparing the genetic and structural analyses of transmission-blocking antigens with pre-erythrocytic and blood-stage antigens, we identify promising P. falciparum vaccine antigens characterized by their conservation with low balancing selection and the presence of infection/transmission-blocking epitopes, which are essential for informing the development of new malaria vaccines. This comprehensive workflow can be adopted for studying the genetic and structural variation of other P. falciparum vaccine targets before developing the next generation of malaria vaccines for effectiveness against natural parasite populations.

Implications of this study Our suggested strategies for designing malaria vaccines include two possible approaches. We emphasize the development of a multi-stage vaccine that combines critical components such as anti-merozoite (Rh5) and transmission-blocking antigens (Pfs25, Pfs28, Pfs48/45, Pfs230). Alternatively, we suggest the creation of transmission-blocking vaccines specifically targeting Pfs25, Pfs28 and Pfs48/45. These innovative approaches show great potential in advancing the development of more potent and effective malaria vaccines for the future.

The authors have declared no competing interest.

This study was funded by Purdue Department of Biological Sciences and Purdue Institute of Inflammation, Immunology and Infectious Disease; Puskas Memorial Fellowship; National Institute of Allergy and Infectious Diseases (U19AI089680).

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The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

The parents or legal guardians provided parental permission for study participants and this study was conducted with the approval of the Biomedical Research Ethics Committee from the University of Zambia (Ref 011-02-18) and from the Zambian National Health Research Authority.

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The raw sequence data are available in the NCBI Sequence Read Archive under BioProject PRJNA932927. Key analysis scripts can be accessed at https://github.com/giocarpi/Pf_capture_div_code.