Malaria poses a huge mortality burden in Sub-Saharan Africa and tropical countries. In 2021, an estimated 241 million infections were reported worldwide, causing about 627,000 deaths (World Health Organization, 2021). This burden was escalated by the COVID-19 pandemic disruptions of existing health systems (World Health Organization, 2021). Despite a declining trend of malaria incidences over the past decade (Rosenthal et al., 2019), pregnant women and children under five still bear the highest burden. P. falciparum, the deadliest among the Plasmodium spp (Murmu et al., 2021), has a complex life cycle alternating between human and mosquito vectors. This, coupled with a lack of effective vaccines, parasite resistance to the available antimalarials, and mosquito resistance to insecticides, hinder efforts to prevent, control and eliminate malaria (Dimala et al., 2018; Cowell and Winzeler, 2019; Maier et al., 2019).

The WHO rolled out the malaria vaccine initiative to support the fight against malaria (Birkett, 2010; Samuels et al., 2022). It is anticipated that a vaccine, in conjunction with other control measures, can synergize and complement each other to eliminate malaria (Dobanõ et al., 2019). One key target for vaccine development is P. falciparum circumsporozoite protein (PfCSP), the major abundant antigen expressed on the surface of sporozoites critical for the invasion of sporozoites in the human liver. Antibodies that target PfCSP have been shown to prevent sporozoite invasion of hepatocytes (Hollingdale et al., 1984; Cohen et al., 2010). The RTS,S (Mosquirix), and R21 are examples of vaccines based on Pfcsp sequence. RTS,S and R21 are currently World Health Organization (WHO)-recommended malaria vaccines for widespread use among children living in malaria-endemic countries (Zavala, 2022).

Although RTS,S and R21 are based on Pfcsp, slight differences exist structurally. RTS,S/AS01 is a sub-unit vaccine comprising of PfCSP region fused with hepatitis B surface antigen (HBsAg), alongside additional unfused HBsAg assembled into virus-like particles (VLPs) which improve antigen presentation to unique T-cell epitopes (Sinnis and Fidock, 2022). Like RTS,S/AS01, R21/Matrix-M combines a VLP expressing PfCSP-HBsAg fusion protein with a saponin-based adjuvant. Unlike RTS,S, the R21 VLP excludes unfused HBsAg, enabling a 1:1 ratio between PfCSP and Hepatitis B antigen (Collins et al., 2017) ( Figure 1 ). Notably, R21 showed at least 75% efficacy over 6 months among Burkina Faso children aged between 5–17 months in phase 2 trials after three rounds of immunization and similar levels of protection even after 1 year of the booster dose (Datoo et al., 2021, 2022). Some reports indicate diminishing effectiveness of vaccines, particularly against P. falciparum strains with diverse Pfcsp alleles to the region used to make the RTS,S vaccine (Neafsey et al., 2015; Pringle et al., 2018; Laurens, 2019). Thus, understanding the role of genetic diversity of Pfcsp has potential implications for vaccine rollout in countries with areas of highly seasonal malaria transmission (Mohamed et al., 2021; Merle et al., 2023).

PfCSP comprises three regions: a conserved N-terminal region, a central repeat region, and a C-terminal region ( Figure 1 ). The N-terminal region contains a conserved KLKQP motif required for hepatocyte entry (Sidjanski et al., 1997). The central repeat region is rich in NANP (Asn-Ala-Asn-Pro) tandem repeats and NVDP (Asn-Val-Asp-Pro) and is recognized as the site for antibody-mediated neutralization. The C-terminal region has two polymorphic Th2R and Th3R sub-regions recognized by CD8+ and CD4+ T-cells respectively (Putaporntip et al., 2009; He et al., 2022) ( Figure 1 ). Natural selection related to host immunity is hypothesized to be responsible for the polymorphism of these sub-regions (Zheng et al., 2019; Huang et al., 2020). The interaction between PfCSP residues and T-cells is mediated by both HLA class I and II expressed on the surface of antigen-presenting cells (APC), which is essential for T-cell immune response. Polymorphisms observed in the Th2R and Th3R region may directly affect the binding affinity of different PfCSP amino acid residues and T-cells, which can enhance or reduce T-cell immune responses. Understanding vaccine candidates’ genetic and immunological interactions is critical in designing effective vaccines. In this study, we assessed the genetic diversity of Pfcsp utilizing paired-end short-read sequences of P. falciparum field isolates from Homabay County, a malaria-endemic region in western Kenya. We further compared our results with parasite populations from two malaria-endemic regions in Kenya using Pfcsp sequences retrieved from the global MalariaGEN database v7.0. Finally, we predicted the binding affinities of 3D7 reference and non-3D7 variants among the major HLA class I and II supertypes to assess their impact on T-cell recognition.

Vaccine development is a crucial aspect of malaria control and elimination. Various antigens have been identified as potential vaccine candidates for malaria, including RTS,S and R21, both based on Pfcsp. A key challenge in malaria vaccine development is the high degree of antigenic diversity due to immune pressure, resulting in different antigenic variants from the reference strain circulating within populations and therefore reducing the effectiveness of current malaria vaccines. This limitation is particularly relevant in endemic malaria regions. It could explain the partial protection reported in RTS,S within the first 12 months and the subsequent waning of the immune response over the next 4 years in children aged between 5 and 17 months (RTS, 2015).

The N-terminal of PfCSP remained relatively conserved across Kenyan isolates, which coincides with other isolates across other regions (Lê et al., 2018; Mohamed et al., 2019; Huang et al., 2020). The N-terminal of CSP binds to heparan sulfate proteoglycans, and cleavage to a conserved region I (KLKQP) by a parasite protease results in to change from being adhesive to invasive to hepatocytes. Specifically, antibodies to the region I found between amino acid ⁸²(DNEKLRKPKHKKLKQPADG)¹⁰⁰ of the N-terminal inhibit the cleavage of CSP and impair invasion both in vivo and in vitro (Espinosa et al., 2015). In the current results, the non-synonymous mutation A98G was observed in high frequency, as previously reported across most global isolates within region I (Huang et al., 2020). The presence of this non-synonymous mutation suggests the area is under selection pressure and may result in structural changes that have an impact on the sporozoite invasion process in the hepatocyte. Despite this, the conserved motif (KLKQP) involved in heparin sulfate binding remained well conserved across Kenyan isolates. The conserved nature of the N-terminal region, coupled with its key functions, collectively makes it an attractive component of the PfCSP vaccine (Bongfen et al., 2009).

The central repeat region in CSP contains species-specific 4-amino acids NANP/NVDP repeats, which make up the part of the immunodominant B-cell epitopes recognized by neutralizing antibodies. Furthermore, the region has been shown to contribute to sporozoite growth in the mosquito. The different number of tetrapeptide NANP repeats was observed across Kenyan samples, contributing to this region’s polymorphisms. The results are consistent with the number of NANP repeats reported across other Kenyan isolates (Lê et al., 2018) and African isolates (Huang et al., 2020). The differing number of repeats has been shown to affect the PfCSP structure. More specifically, Escalante et al. reported that the stability of type-β PfCSP is increased with the number of repeats (Escalante et al., 2002). The current RTS, S vaccines have 19 NANP tetrapeptide repeats, and to date, there is no direct evidence linking the number of NANP repeats to vaccine efficacy. Nonetheless, targeting regions with repeat regions has been shown to result in less durable antibody responses. Repeat regions such as NANP have been suggested to be decoy regions used by the parasite to prevent the immune system from mounting a strong neutralizing effect.

A high level of polymorphism was observed within the Th2R and Th3R regions of the C-terminal of PfCSP. The Th2R region was more polymorphic compared to the Th3R regions, as previously reported (Amegashie et al., 2020; Dieng et al., 2023). Considering ThR2 and ThR3 regions are involved in the CD4+ and CD8+ T cell responses, the presence of polymorphisms may help the parasite escape the immune pressure from the host, which has a direct impact on the overall efficacy of the vaccine. These results are similar to previous reports, which showed between one and six changes in amino acid occurring within the Th2R and Th3R regions of Zambia, Ghana, and DRC populations (Pringle et al., 2018).

The weak positive Tajima’s D values reported in the C-terminal region of Pfcsp across the three different sites in Kenya which could suggest balancing selection primarily due to host immune pressure on the C-terminal. Nonetheless, the positive Tajima’s D values across the three sites were non-significant and therefore inconclusive. Evidence of balancing selection has previously been reported across isolates from Navirongo in Ghana and populations from Malawi (Bailey et al., 2012; Amegashie et al., 2020). The weak positive Tajima’s D values could be further explained by the fact that Homabay, Kisumu, and Kilifi are regarded as malaria endemic regions within Kenya, and therefore, raising the possibility that the variants could be maintained due to their selective advantage, such as conferring better immune response against malaria or being resistant to malaria. The overall values of haplotype diversity for the C-terminal were also high, suggesting a higher level of genetic diversity across Kenyan isolates which is a common feature of most African isolates (Huang et al., 2020). Further analysis of the nucleotide diversity was similar to other global isolates, showing a relatively similar plot composed of high genetic variation within the Th2R and Th3R regions.

Haplotype analysis of the C-terminal region revealed a branched pattern in terms of haplotype diversity. In addition, a total of 42 singletons were identified, suggesting Kenyan isolates show high levels of genetic diversity similar to other African isolates. The 3D7 reference genome used in the development of the RTS,S, and R21 vaccines shared the highest frequency with most isolates from Kisumu and Kilifi but not from Homabay. The genetic heterogeneity might result in different levels of vaccine efficacy, especially in areas where the vaccine haplotype was absent (Pringle et al., 2018). Therefore, consistent monitoring of the existing haplotypes while administering vaccines that target the C-terminal region of CSP will be valuable, especially in high transmission areas. In addition, developing specific individual vaccines that target the most prevalent haplotype in malaria-endemic regions is another solution.

HLA class I and II typically exhibit different binding affinities among diverse populations. Polymorphisms in specific regions recognized by HLA alleles can either provide protection against a variety of pathogens or increase susceptibility to diseases. The strength of binding between antigenic peptides and the groove of the HLA, as well as with T-cell receptor peptides, determines TCR activation, signaling, and cell responsiveness (Baumgaertner et al., 2022). Given that regions Th2R and Th3R are crucial for evoking CD4+ and CD8+ responses, examining their interaction with HLA alleles offers insights into the impact of existing variants on T-cell responses. Our predicted results indicate that multiple haplotypes exhibit better binding affinity compared to the reference strain 3D7 for both HLA class I and II supertypes. These findings align with observations from the evaluation of Ghanian isolates. In that context, the 3D7 reference clearly bound to the HLA class II allele, suggesting that the reference might be binding to additional epitopes present in PfCSP, which were not assessed in this study (Dieng et al., 2023).

The samples used in this study were collected from Homabay in Kenya, one of the malaria-endemic regions in Kenya. The different malaria endemic regions have unique parasite transmission diversities that may continuously impact CSP diversity. To understand the complete CSP diversity in Kenya, it is important to assess mutations across the country continuously. Also, the study only utilized the major HLA class I and II supertypes present globally, and might not provide a representative picture of Kenyan HLA class I and II supertypes since which are are currently not resolved especially among malaria endemic regions. Other factors such as that may influence HLA variation and T-cell responses, such as exposure differences, T-cell receptor variation, and vaccination status, were not examined. Lastly, the sequenced isolates used in analyzing the current data were cultured in vitro and therefore, there is also possibility of some mutations being introduced during the culturing process.

This study provides essential information on the genetic diversity and polymorphism of Pfcsp from Kenya. The N-terminal region and central repeat region within the Kenyan isolates remained relatively conserved, while the Th2R and Th3R in the C-terminal region showed high levels of polymorphism. Lastly, the 3D7 reference did not bind weakly or strongly to supertype alleles, although multiple different haplotypes were able to be recognized by the same HLA alleles.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material .

The studies involving humans were approved by Mount Kenya University Ethics and Research Review Committee (MKU-ERC). The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants’ legal guardians/next of kin. The animal study was approved by Mount Kenya University Ethics and Research Review Committee (MKU-ERC). The study was conducted in accordance with the local legislation and institutional requirements.

MM: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. SM: Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing – review & editing. JK: Data curation, Formal analysis, Investigation, Methodology, Software, Visualization, Writing – review & editing. HW: Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – review & editing. DK: Data curation, Investigation, Supervision, Validation, Visualization, Writing – review & editing. BK: Conceptualization, Data curation, Formal analysis, Investigation, Supervision, Validation, Writing – review & editing. JG: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Writing – review & editing.