The study enrolled 135 individuals from the endemic area of Plasmodium vivax malaria in Porto Velho, Rondônia State in the Amazon region of Brazil. Of these, 117 individuals had their infection diagnosis confirmed by positive blood smears, while 18 individuals did not have acute malaria and were considered exposed. Additionally, 153 individuals residing in a non-endemic area for malaria in the municipality of Conde, Paraíba state, Northeastern Brazil, who had experienced an outbreak of P. vivax malaria, were included. Of the total number, 17 individuals had their malaria diagnosis confirmed by blood smears, while 136 individuals did not have the infection and were considered exposed. The blood samples from the individuals affected by the outbreak were collected in July of 2019. As a negative control, sera from 15 healthy individuals living in non-endemic areas of the Brazilian state of Minas Gerais were collected. All participants were over 18 years of age and provided informed consent to participate in the study.

This study was conducted following the ethical standards of the Research Ethics Committee of the Oswaldo Cruz Foundation (FIOCRUZ) in Pernambuco, and by the Universidade Federal de Minas Gerais (UFMG), Brazil. The study was approved by the Research Ethics Committee of UFMG under protocol CAAE: 27466214.0.0000.5149, and by the Research Ethics Committee of the FIOCRUZ under protocol CAAE: 15891619.1.0000.5190.

Peptide arrays corresponding to the entire protein PvVIR14 were synthesized on a nitrocellulose membrane, as previously described (Siqueira et al., 2023), and probed using a pool of serum from healthy subjects (Negative Control, NC), and in a second moment with serum from patients with active P. vivax (Positive Control, PC). Peptides that demonstrated reactivity in both the NC and PC groups were excluded from further analysis, according to the previously established methodology (Bueno et al., 2011; Fantin et al., 2021).

The immunoblotting assay was conducted as previously described (Siqueira et al., 2023), with minor alterations. In brief, the peptide’s ability to recognize specific IgG antibodies was evaluated using a pooled serum sample (dilution 1:500) from 10 individuals with active P. vivax infection (PC), residing in an endemic region. For detection, a secondary anti-human IgG antibody was applied at a 1:10.000 dilution. As NC, a pooled serum sample from 10 healthy individuals from non-endemic, malaria-free areas was utilized. The quantification of spot signal intensities was conducted using the ImageQuant LAS 4000 digital imaging system software, with the analyses performed using ImageJ.

The densitometric value of each spot was determined and normalized using ImageJ, as previously described (Siqueira et al., 2023). After the membrane analysis, eight peptides were able to be synthesized in their soluble form using the solid-phase peptide synthesis (SPPS) technique on a 10 µmol scale using the ResPep SL automated synthesizer (Intavis) as previously described (Ruas et al., 2025). The eight successfully synthesized peptides then proceeded for subsequent immunogenicity analyses.

To determine the presence of specific IgG and IgM antibodies against PvVir14 and its derived peptides, an Enzyme-linked Immunosorbent Assay (ELISA) was performed as previously described (Fantin et al., 2023). This assay was carried out using serum from individuals from both endemic and non-endemic areas of P. vivax malaria. Sera from individuals residing in non-endemic areas and who had never experienced malaria were utilized as negative controls. The cut-off values were calculated as previously described (Fantin et al., 2021; Fantin et al., 2023), and the samples were considered positive when Reactivity Index (R.I.) ≥1 (Bueno et al., 2011).

The depletion ELISA was performed to evaluate the influence of peptides (L06, H08. And G08) on the recognition of PvVir14 by specific IgG antibodies, following the methodology earlier described by (Santiago et al., 2011). In brief, 96-well plates were coated with 2 µg/well of each analyzed peptide and incubated overnight at 4°C. Subsequently, the plates were washed and blocked under the protocol. The diluted sera (1:100) were then added and incubated overnight at 37°C. On the following day, the sera were transferred to 96-well plates previously coated overnight with PvVir14 (0.5 µg/well) after washing and blocking. The ELISA was then conducted by the previously described methodology.

All data presented here was analyzed using GraphPad Prism 8.0 (GraphPad, San Diego, USA). The normality distribution of all data was analyzed by the Shapiro-Wilk test. Kruskall-Wallis followed by Dunn’s post-test was performed. Fisher’s exact test was used to determine the equality of variances among normal populations. The receiver operating characteristic (ROC) curve was employed to analyze the specificity and sensitivity of PvVir14 and derived peptides. All data were considered statistically significant when p < 0.05.

The study involved 135 participants from an endemic area of P. vivax malaria in Brazil, with a gender distribution of 31.86% female (n=43) and 68.14% male (n=92). The average age was 54 years (± 17.28) for females and 46 years (± 13.62) for males. Regarding previous malaria infections, 21.48% (n=29) had no history of malaria, 20% (n=27) had 1-2 previous episodes, and 58.52% (n=79) had three or more episodes ( Table 1 ).

The densitometry results were analyzed ( Table 2 ), and the eight peptides successfully synthesized (identified as H08, K09, I03, G08, F08, H04, L06, and N04) were found to be recognized by specific IgG antibodies present in the sera of individuals from endemic areas for P. vivax malaria. The frequency of individuals that are positive for the peptides H08, G08, and L06 was found to be significantly higher than that of the others (p<0.0001, Fisher’s exact test) ( Figure 1A ). It was observed that 70.8% of the individuals exhibited reactivity to the PvVir14 whole protein alone. Concerning the synthesized peptides, H08, G08, and L06 demonstrated a frequency of 63.4%, 49.2%, and 72.6% reactivity in the sera, respectively. When the three peptides were pooled together, the reactivity frequency increased to 81.2% ( Figure 1B ).

After analyzing the frequency of IgG reactivity among individuals from the endemic area, we classified the population into two different categories: (i) with acute malaria infection and compared to (ii) non-infected but exposed to malaria participants. Specific IgM reactivity to PvVir14, L06, H08, and G08 was observed in 27.3%, 22.2%, 10.2%, and 14.5% of P. vivax-infected individuals, respectively ( Figure 2A ). The frequency of total IgG reactivity was higher among infected individuals, with 72.2% (p<0.0001), 80% (p=0.0025), 68.1% (p=0.0104), and 58.6% (p=0.0008) for PvVir14, L06, H08, and G08, respectively ( Figure 2B ).

The individuals from the endemic area enrolled in the study were then organized into three groups based on their malaria history: (1) Those who were exposed but never exhibited the acute infection, (2) those who experienced their first malaria episode, and (3) those who had two or more cases of malaria. The IgM reactivity for PvVir14 was 33.3% and 29.3% for the first and third groups, respectively ( Figure 3A ). Among individuals experiencing their first episode of malaria, the frequency of IgM reactivity was found to be 33.3% for the peptides L06 and G08, and 9.5% for the peptide H08 ( Figure 3A ).

The IgG reactivity against PvVir14 and the peptides was higher in individuals with the first or multiple cases of malaria ( Figure 3B ). Anti-PvVir14 IgG reactivity had a frequency of 75.2% in individuals with multiple cases of malaria, while those in the first episode of the disease displayed a frequency of 66.7% (p<0.0001, Fisher’s exact test). Individuals experiencing their first episode of malaria had higher IgG reactivity against the peptides L06 (79%, p<0.0001), and G08 (71.4%, p=0.0003), when compared to those with no previous malaria history, and those with multiple cases of infection. For individuals in their first malaria episode and those with multiple cases of malaria, the frequency of IgG reactivity to the peptide H08 was 65.2%, and 65%, respectively ( Figure 3B ).

Considering the 153 individuals from a non-endemic area for Malaria, who experienced the outbreak, 56.35% (100) were female with a median age of 35 years; the male individuals (34.65%) had a median age of 43 years. In terms of previous malaria episodes, 84.31% had no prior infections, 13.07% had experienced 1 to 2 previous infections, and 2.61% had experienced 3 or more previous infections. Additionally, 13.72% (21) of the individuals had previously visited a malaria-endemic area, while 86.27% (132) had not ( Table 3 ).

The highest IgM reactivity was found for the peptides H08 (61%) and G08 (68.6%) ( Figure 4A ). Regarding the frequency of anti-PvVir14 IgG, 20.3% of the participants had circulating antibodies. In contrast, the frequency of antibodies for the three peptides was lower than 10% ( Figure 4B ). Within the individuals with acute infection, the frequency of IgM against PvVir14, and anti-L06, -H08, and -G08 was 29.4%, 47%, 88.2%, and 82.3%, respectively ( Figure 5A ). Specific anti-PvVir14 IgG reactivity presented a frequency of 47% within infected individuals, while IgG against the peptides L06 and H08 was equal (17.6%), for the peptide G08 displayed 0% of reactivity for the same group ( Figure 5B ).

In the endemic area, peptides L06, H08, and G08 exhibited similar sensitivity and specificity values exceeding 80% when compared to PvVir14 values for infected individuals against healthy controls ( Supplementary Figure S1A ). Nevertheless, L06 was the most effective peptide for identifying infected individuals ( Table 4 ). When analyzing the ability of the protein and peptides to differentiate infected individuals from exposed individuals, PvVir14 demonstrated sensitivity and specificity values exceeding 90% ( Table 4 ). Only the peptide H08 was able to discriminate between infected individuals from exposed ones, with sensitivity and specificity values of 61.1% and 72.7%, respectively ( Supplementary Figure S1B ; Table 4 ).

To analyze the influence of peptides over the PvVir14 recognition by specific IgG, a depletion ELISA was performed. The results indicated that the peptides L06, G08, and H08 exhibited immunodominance over the protein, as evidenced by a reduction in reactivity of 54.5%, 95.7%, and 88.5%, respectively ( Figure 6A ). This was further corroborated by the pronounced decline in IgG titres that recognized PvVir14 following depletion ( Figure 6B ). Specifically, prior to depletion, the mean optical density (OD) values for PvVir14 were 0.6980 (± 0.3577), 0.6600 (± 0.3095), and 0.7106 (± 0.3171), reflecting the baseline recognition of PvVir14 by IgG antibodies. After depletion using peptide L06, the mean OD decreased to 0.3270 (± 0.2294), while depletion with peptides H08 and G08 reduced the mean OD to 0.2681 (± 0.3035) and to 0.2995 (± 0.2597), respectively. These results demonstrate that while all three peptides contribute to PvVir14 recognition, the peptides H08 and G08 exhibit the highest level of immunodominance, significantly diminishing the protein recognition by specific IgG.