This study was conducted in two regions of Tanzania, Geita in the northwest and Kigoma in the west, where malaria prevalence was reported at 13% in both regions in 2022 (21). Based on high Plasmodium falciparum endemicity, two districts were randomly selected from each region, Nyang’hwale and Chato in Geita, and Kibondo and Kasulu in Kigoma. Subsequently, two villages were randomly selected from each district ( Figure 1A ). In total, eight villages from high-endemic micro-geographical areas were included in the study.

Peripheral blood samples were collected between December 2022 and July 2023 from participants residing in the selected villages. The clinical sample collection procedures were described previously (21). Briefly, whole blood was obtained from asymptomatic individuals infected with P. falciparum during household visits conducted by the research team. After obtaining informed consent, demographic information was recorded using a standardized questionnaire. Individuals were classified as asymptomatic if they had no fever, no malaria-related symptoms in the previous 4–5 days, had not taken any antimalaria drugs within the past seven days, and had a positive microscopic diagnosis for parasitemia at any level.

Malaria infection was initially assessed using the Bioline™ Malaria Ag P.f/Pan rapid diagnostic test (RDT) (Abbott, Chicago, IL, USA). Thin blood smears were prepared by two trained microscopists, stained with Giemsa, and examined under a light microscope. Parasitemia was determined by counting parasites per 200 white blood cells, in accordance with the WHO Malaria Microscopy Standard Operating Procedures. As per national malaria treatment guidelines in Tanzania, asymptomatic individuals who tested positive for P. falciparum were treated with artemether-lumefantrine.

Following diagnosis, serum was separated and stored at -20 °C at a nearby health facility laboratory, while whole blood was preserved as dried blood spots (DBS) on Whatman 3MM filter paper. The serum and DBS samples were later transported to Kangwon National University for molecular diagnosis by qPCR, pfeba-175 RII gene sequencing, and antigenicity assessment.

A total of 172 whole blood samples were collected and processed for humoral immune response screening by protein microarray. However, only 122 (70.9%) samples yielded high-quality pfeba-175 RII sequences due to low parasitemia and suboptimal DBS quality. For negative controls in the protein microarray, healthy serum samples (n = 64) were obtained from children under 10 years of age residing in non-endemic regions of the Republic of Korea during routine health examinations at Kangwon National University Hospital.

Genomic DNA was extracted from a single dried blood spot (50 μL) using the QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany), following the manufacturer’s instructions, with a final elution volume of 50 μL. Extracted DNA was stored at -20 °C until use.

The pfeba-175 RII gene was successfully amplified and sequenced from 122 samples collected from high-endemic villages in Tanzania. PCR was performed to amplify the target region for downstream sequencing and analysis. Target-specific primers covering the entire coding region were designed based on the pfeba-175 RII gene (PF3D7_0731500) ( Supplementary Table S1 ). Each 20 µL PCR reaction contained KOD One PCR Master Mix (Toyobo, Osaka, Japan), 1 µL of genomic DNA, 16 µL of distilled deionized water (DDW), and 1 µL of each 10 μM primer. The thermal cycling conditions were as follow, initial denaturation at 98°C for 7 minutes, 45 cycles of 98°C for 10 seconds (denaturation), 60°C for 5 seconds (annealing), and 68°C for 10 seconds (extension), followed by a final extension at 68°C for 10 minutes. Amplicons were visualized by electrophoresis on a 1.2% agarose gel and purified using a PureLink™ PCR Purification Kit (Invitrogen, Waltham, MA, USA). The purified amplicon was subjected to Sanger sequencing using the PCR forward primer and sequencing reverse primer on an ABI3730xl DNA analyzer (Nbit, Chuncheon, Republic of Korea) ( Supplementary Table S1 ). Full-length pfeba-175 RII gene sequences were assembled using SnapGene v2.3.2 and the Lasergene 11 package, following chromatogram inspection for quality validation. The rare and novel genetic variants were confirmed by three independent sequencing experiments. The complete pfeba-175 RII sequences have been deposited in GenBank (accession numbers: PV683895 - PV684016).

The average number of nucleotide differences per site (π) was calculated to assess the genetic diversity of the pfeba-175 RII gene. Sequence diversity within the population was evaluated based on the number of polymorphic sites, number of haplotypes, and haplotype diversity (Hd) were computed using DnaSP v5.10 (28). To investigate potential signatures of natural selection, several neutrality tests were conducted, including Tajima’s D, Fu and Li’s D* and F*, and Fu’s Fs. Under the neutral theory of evolution, Tajima’s D is expected to be zero. Significantly negative values may indicate recent population expansion or purifying selection, while significantly positive values suggest balancing selection or a population bottleneck (29). Fu and Li’s D* and F* tests further assess deviations from neutrality, where significantly negative values typically indicate recent population growth with an excess of rare alleles, whereas significantly positive values suggest population contraction or the influence of background selection (30). Fu’s Fs test assesses demographic expansion by evaluating haplotype distributions. Negative Fs values indicate an excess of rare haplotypes, which may result from recent population expansion or genetic hitchhiking (31). To further explore selection pressure, the number of synonymous substitutions per synonymous site (d_S) and nonsynonymous substitutions per nonsynonymous site (d_N) were calculated using the Nei and Gojobori method with 1,000 bootstrap replicates in Mega 11 software. A d_N-d_S greater than zero suggests positive selection, while less than zero suggests purifying selection. In addition, the McDonald-Kreitman (MK) test was performed to compare within-species polymorphisms with between-species divergence. For this analysis, P. reichenowi EBA-175 RII (PRCDC_0728300), a member of the Laverania clade closely related to P. falciparum, was used as an outgroup. DnaSP v5.10 was used to perform the MK test and generate relevant summary statistics, including the neutrality index (NI), which further supports inference of selection. Haplotype clustering patterns were visualized using the median-joining method in Network 10.1, while haplotype diversity was analyzed using DnaSP v5.10.

The tertiary structure of PfEBA-175 RII was retrieved from the Protein Data Bank (PDB ID: 1ZRO), which represents the PfEBA-175 RII crystallized in the presence of α2, 3-sialyllactose (6). Non-synonymous mutations were mapped and visualized onto the structure using Chimera X 1.9, with particular attention to those located at or near the interaction interface between PfEBA-175 RII and the host receptor.

The recombinant PfEBA-175 RII (amino acids 152-743, PF3D7_0731500) was codon-optimized for mammalian expression, corresponding to a molecular weight of approximately 81.2 kDa. The gene was amplified using gene-specific cloning primers ( Supplementary Table S1 ) and cloned into the pTT5 mammalian expression vector with a C-terminal 6x His tag using the In-Fusion^® HD Cloning Kit (Clontech, Mountain View, CA, USA). Expression was performed in HEK293-6E cells via transient transfection using linear polyethylenimine hydrochloride (PEI). After 5 days of culture at 37°C in a humidified incubator with 5% CO₂, the secreted recombinant protein in the culture supernatant was harvested. The recombinant protein was purified using Ni-NTA agarose resin (QIAGEN) and eluted with buffer containing 300 mM imidazole, 50 mM HEPES, 5% glycerol, and 150 mM NaCl. Purity was assessed by SDS-PAGE (4-12% Bis-Tris gradient gel, Invitrogen) and Coomassie Brilliant Blue staining (Sigma-Aldrich, St. Louis, MO, USA).

Protein microarray analysis was performed to evaluate the antigenicity of PfEBA-175 RII. Three-aminopropyl coated glass slides were prepared as previously described (25). PfEBA-175 RII was printed onto each slide at an optimal concentration of 25 ng/µL per spot, followed by incubation for 2 hours at 37°C. After incubation, the slides were blocked for 1 hour at 37°C with 5% BSA in PBS-T (0.1% Tween-20). Sera from asymptomatic P. falciparum-infected individuals and healthy controls were diluted 1:25 in PBS-T and applied to each slide in duplicate for 1 hour at 37°C. IgG reactivity was visualized using 10 ng/µL of Alexa Fluor 546-conjugated goat anti-human IgG (Invitrogen) in PBS-T for 1 hour at 37 °C. Array slides were scanned using the InnoScan 300 scanner (Innopsys, Carbonne, France). The threshold for a positive response was defined as the mean fluorescence intensity (MFI) of healthy control plus two standard deviations.

Mean fluorescence intensity (MFI) values associated with distinct amino acid polymorphisms at each residue were quantitatively aggregated and represented as a heatmap. The resulting data matrix was organized with amino acid substitutions as rows and the corresponding residue positions as columns. Visualization employed hierarchical structuring of antigenic variation patterns using the seaborn Python package with the rainbow colormap. Annotated mean MFI values within each cell enabled precise assessment of immune reactivity differences driven by specific amino acid changes.

Group comparisons for protein microarray data were performed using unpaired Student’s t-tests. Associations between antigenicity and demographic variables (gender, age, parasitemia) were assessed by Spearman correlation (ρ). A p-value < 0.05 was considered statistically significant. Data were analyzed and visualized using SigmaPlot v12.0 (Systat Software Inc., San Jose, CA, USA) and GraphPad Prism v8 (GraphPad Software, San Diego, CA, USA) software.

The National Institute for Medical Research (NIMR), a branch of Tanzania’s Ministry of Health (MoH), provided ethical guidelines and approved methods for the collection of all clinical samples (NIMR/HQ/R.81/Vol.IX/4114). The Kangwon National University Institutional Review Board approved the secondary use of clinical samples for further processing and experimental protocols (KWNUIRB-2022-06-008). Informed consent was obtained in writing from all adult participants, whereas for child participants, consent was secured from their parent or legal guardian.

This study involved collecting Plasmodium falciparum isolates from asymptomatic participants in the Geita and Kigoma regions of Tanzania, both of which were identified as high malaria burden in 2022 (21). Geita contributed 53.5%, comprising Chato 24.4% (GCR 12.8%; GCI 11.6%) and Nyang’hwale 29.1% (GNK 14.0%; GNN 15.1%), whereas Kigoma contributed for 46.5%, with contributions from Kasulu 27.3% (KKM 13.4%; KKN 14.0%) and Kibondo 19.2% (KKB 8.1%; KKK 11.1%) ( Figure 1A ).

Parasitemia levels and demographic factors were compared across asymptomatic individuals ( Supplementary Table S2 ). No significant differences in parasitemia were observed between children under 5 years of age and those aged 6–10 or over 25 years. In contrast, parasitemia was significantly higher in the 11-15, 16-20, and 21-25-year age groups compared to children under 5 years ( Figure 1B ). Regional comparisons revealed significant variation in parasitemia levels. Kumuhasha (KKK) village exhibited a significantly higher mean parasitemia (Mean ± S.D., 25,244 ± 42,248 parasites/µL) than all other study villages except Nyangalamila (GNN) ( Figure 1C ). Although other regions showed lower mean parasitemia levels with considerable variability, no statistically significant differences were observed among them.

The pfeba-175 gene (PF3D7_0731500) spans 4,509 base pairs, consists of four exons, and is located on chromosome 7. Region II (RII) comprises 1,776 base pairs encoding 592 amino acids, with a predicted molecular weight of approximately 81.2 kDa ( Figure 2A ). Nucleotide diversity (π) analysis of the RII region revealed that the polymorphic sites were evenly distributed across all domains ( Figure 2B ). The tertiary structure of PfEBA-175 RII was obtained from the Protein Data Bank (PDB ID: 1ZRO), and key structural features were visualized using a color map, highlighting the EBL domains (F1 and F2), glycan-binding sites, dimer interface residues, and B-cell epitopes recognized by the R217 and R218 monoclonal antibodies (32) ( Figure 2C ). Within the RII region, two synonymous mutations were identified at I401I (c.ATT>ATA, 0.8%) and I705I (c.ATA>ATC, 1.6%), and two deletion mutations were observed at I401 (21.3%) and S402 (21.3%) ( Supplementary Table S3 ). The positions of deletion mutations were excluded from further analysis of natural selection. In total, 27 non-synonymous mutations were detected in the RII region, with high population frequencies (>50%) for K279E (65.6%), P390S (59.8%), and R664S (55.7%) ( Figure 2C ). Among these non-synonymous mutations, Q584E/K (17.2%/27.9%) represented multiple allelic substitutions. Additionally, four novel non-synonymous mutations, T198K (1.6%), A271D (0.8%), K448N (0.8%), and D619H (0.8%) were identified. Several non-synonymous mutations were located in the F1 domain, including T198K, K226E, A271D, E274K, I275K, K279E, K286E, and D336Y. Additional mutations in the F2 domain included N577K, Q584E, and E592A, D619H ( Figure 2C ; Supplementary Table S3 ). None of the mutations were located within the glycan-binding residues. Importantly, mutations at K478N (16.4%), K481I (26.2%), and L482V (3.3%) were found within the R217 epitope, which is possibly implicated in invasion inhibition. Among them, L482V was mapped to a region overlapping the R217 epitope and the dimer interface. Additionally, N577K (13.9%) was also located at the dimer interface ( Figure 2C ; Supplementary Table S3 ).

Sequence analysis of the pfeba-175 RII gene from 122 clinical isolates revealed 26 polymorphic sites and a total of 27 mutations. These included a non-synonymous substitution at position 584 (Q584E/K), where multiple amino acid changes were observed at a single site ( Table 1 ). The average of overall nucleotide diversity (π) in the study population was revealed 0.00359 ± 0.00012 (π ± S.D.). Based on the region, Geita and Kigoma also showed similar nucleotide diversity (π) that 0.00354 ± 0.00019 and 0.00365 ± 0.00015, respectively ( Table 1 ). Additionally, comparable levels of nucleotide diversity (π) were observed at the district level from 0.00348 ± 0.00028 (Nyang’hwale) to 0.00369 ± 0.00026 (Chato). And at the village level from 0.00319 ± 0.00071 (KKB) to 0.00394 ± 0.00032 (GCR) ( Table 1 ). At the micro-geographical scale, nucleotide diversity was consistently high and relatively uniform across villages.

Neutrality tests were conducted to assess whether natural selection has shaped the polymorphism observed in the pfeba-175 RII gene. Intra-species analysis showed weakly positive values for Tajima’s D (0.78498), Fu and Li’s D* (0.40105), and Fu and Li’s F* (0.66211), which may suggest balancing selection or population structure within the species ( Table 1 ). However, none of these values were statistically significant, indicating no strong deviation from neutrality. In contrast, Fu’s Fs (-30.614) showed a highly negative value, providing strong evidence for recent population expansion ( Table 1 ). To further assess selection pressure, a Z-test comparing the rates of non-synonymous and synonymous substitution (d_N–d_S = 3.15, p < 0.001) revealed a significant excess of non-synonymous changes, consistent with action of positive selection ( Table 2 ). Inter-species analysis using the McDonald–Kreitman (MK) test with P. reichenowi as an outgroup revealed significant purifying selection (NI = 11.258, p = 0.003125) ( Table 2 ). Taken together, these results suggest that pfeba-175 RII has likely undergone recent population expansion and may be subject to positive selection acting on non-synonymous sites across multiple alleles within P. falciparum. While the gene appears to be under functional constraint between species, it may be evolving under different selective pressures within the species.

A total of 51 distinct haplotypes were identified from the amino acid sequences of 122 clinical isolates, demonstrating substantial genetic diversity within the pfeba-175 RII gene ( Table 1 and Figure 3 ). This diversity was largely attributed to a high number of singleton haplotypes (n = 32), which supports the scenario of recent population expansion. The remaining 19 haplotypes were shared among multiple isolates and exhibited geographically mixed distributions, suggesting gene flow across different regions. At the village level, Mugombe (KKM) exhibited the highest haplotype diversity, with 14 isolates represented by 13 distinct haplotypes, whereas Bunyambo (KKB) showed the lowest diversity, with six haplotypes ( Figure 3 ). The novel mutation T198K (H02), A271D (H41), K448N (H11), and D619H (H20) were identified in specific haplotypes originating from villages, including Nyangalamila (GNN), Kayenze (GNK), Kumuhasha (KKK), and Nyamnyusi (KKN), respectively ( Figure 3 ). In addition, three non-synonymous substitutions within the R217 monoclonal antibody epitope such as K478N (16.4%), K481I (26.2%), and L482V (3.3%), observed in isolates from diverse geographic locations. Especially, K478N substitution was detected independently in isolates from H32 (GCI), H06 (GNN), H29 (GCI), and showed a contiguous spread originating from H48 (KKM) ( Figure 3 ). The K481I mutation, which frequently co-occurred with K478N, also appeared to have spread contiguously from H48 (KKM). In contrast, the L482V substitution exhibited an independent distribution pattern, occurring in isolates from H09 (GNN), H27 (GCI), H30 (GCI), and H40(KKN) ( Figure 3 ).

To assess humoral immune responses, recombinant PfEBA-175 RII (81.2 kDa) was expressed in a soluble form and used to screen sera from asymptomatic individuals ( Figure 4A ). Each serum sample was tested in duplicate wells, and fluorescence signals were averaged to obtain the mean fluorescence intensity (MFI). Total IgG reactivity against PfEBA-175 RII was significantly higher in asymptomatic individuals (MFI ± S.D., 11,526 ± 11,707) compared to healthy controls (1,384 ± 573.3) ( Table 3 ). Overall assay sensitivity for detecting PfEBA-175 RII–specific IgG in asymptomatic individuals was 84.8% (95% CI, 78.7–89.4), and specificity in healthy controls was 92.1% (95% CI, 82.9–96.6)( Table 3 ). When stratified by region, IgG response was similar between Kigoma (85.0%) and Geita (84.7%) ( Table 3 ). However, at the village level, the seropositivity varied substantially. Most villages showed over 90% sero-positivity, whereas Bunyambo (KKB) and Kayenze (GNK) exhibited notably lower IgG responses at 50.0% and 58.3%, respectively ( Figure 4B ; Table 3 ). Analysis of demographic variables revealed no significant differences in IgG responses between gender or parasitemia (ρ = 0.041, p = 0.595) ( Figures 4C, D ). In contrast, a significant positive correlation was observed with age (ρ = 0.333, p < 0.001), suggesting that antibody response may be strengthened with repeated exposure over time ( Figure 4E ).

To evaluate the impact of sequence variation on antibody recognition, mean fluorescence intensity (MFI) values were compared across various amino acid substitutions at multiple positions ( Figure 4F ). Several substitutions resulted in marked reductions in MFI, suggesting potential involvement in antibody binding. Notable examples include D168H, K448N, T198K, and I275K in the F1 domain, as well as D619H in the F2 domain. At 168 and 619, substitution of aspartic acid (MFI = 11,488 and 11,484, respectively) with histidine (2,307 and 2,796, respectively) resulted in dramatic decreases in MFI, indicating the importance of these residues in maintaining epitope integrity ( Figure 4F ). These findings imply that negatively charged residues at these sites may play a structural or electrostatic role critical for antibody binding. Similarly, the substitution of threonine with lysine at position 198 and isoleucine with lysine at position 275 led to a notable reduction in MFI, suggesting that charge alterations at these positions disrupt antibody recognition. In contrast, at positions 226, 271, 279, 388, 390, 403, and 577, the introduction of a positive charge enhanced antibody binding, indicating that in certain contexts, charge addition can be beneficial. This further supports the importance of charge alterations at specific positions for antibody recognition on PfEBA-175 RII ( Figure 4F ).

Many other positions tolerated substitutions without substantial changes in MFI, suggesting limited involvement in direct antibody contact, including the R217 epitope ( Figure 4F ). Collectively, these findings highlight key residues that contribute to the antibody binding interface, either through direct interaction or by stabilizing the structural conformation necessary for effective antibody binding.