Identification and validation of a novel panel of Plasmodium knowlesi biomarkers of serological exposure

Abstract

Background: Plasmodium knowlesi is the most common cause of malaria in Malaysian Borneo, with reporting limited to clinical cases presenting to health facilities and scarce data on the true extent of transmission. Serological estimations of transmission have been used with other malaria species to garner information about epidemiological patterns. However, there are a distinct lack of suitable serosurveillance tools for this neglected disease.

Methodology/principal findings: Using in silico tools, we designed and expressed four novel P. knowlesi protein products to address the distinct lack of suitable serosurveillance tools: PkSERA3 antigens 1 and 2, PkSSP2/TRAP and PkTSERA2 antigen 1. Antibody prevalence to these antigens was determined by ELISA for three time-points post-treatment from a hospital-based clinical treatment trial in Sabah, East Malaysia (n = 97 individuals; 241 total samples for all time points). Higher responses were observed for the PkSERA3 antigen 2 (67%, 65/97) across all time-points (day 0: 36.9% 34/92; day 7: 63.8% 46/72; day 28: 58.4% 45/77) with significant differences between the clinical cases and controls (n = 55, mean plus 3 SD) (day 0 p<0.0001; day 7 p<0.0001; day 28 p<0.0001). Using boosted regression trees, we developed models to classify P. knowlesi exposure (cross-validated AUC 88.9%; IQR 86.1-91.3%) and identified the most predictive antibody responses.

Conclusions/significance: The PkSERA3 antigen 2 had the highest relative variable importance in all models. Further validation of these antigens is underway to determine the specificity of these tools in the context of multi-species infections at the population level.

Fig 1. Flowchart summarising the experimental strategy used in the identification and validation of the P. knowlesi-specific candidates.

Fig 2. Plasmodium knowlesi-specific recombinant antigen constructs.

Schematic representations for each protein are shown with key features labelled. (a) PkSERA3 shows the location of the putative pro-enzyme and enzyme domains. The predicted subtilisin (SUB) 1 cleavage sites in relation to variable regions 1 and 2 and the cytoplasmic domain [29]. (b) PkSSP2/TRAP contains a von Willebrandt A domain (vWA), thrombospondin type (TSP) 1 motif, a C-terminal transmembrane (TM) region and a cytoplasmic terminal domain (CTD). Putative T-cell and B-cell epitopes are highlighted with an asterix or black square, respectively [30]. (c) PkTSERA2 shows the lack of central enzyme domain due to truncation of the sequence [29]. Predicted secondary structures generated in I-Tasser [31] are shown above each scheme. Red boxes represent helices, blue arrows sheets and the black line coils. The position of recombinant proteins are highlighted below each scheme with the N- and C-terminal amino acid positions indicated. The overall length of each protein is referenced by the amino acid ruler above each secondary structure prediction. For all proteins SP refers to the signal peptide.

Fig 3. SDS-PAGE of purified recombinants.

Lane 1: SERA3 ag1; Lane 2: SERA3 ag2; Lane 3: SSP2/TRAP and Lane 4: TSERA2 ag1. Band sizes are indicated on one side in kDa (Fermentas PageRuler Prestained Protein Ladder). Samples were ran under reducing conditions at approximately 0.4 mg/ml per lane on a 4–20% gradient gel (NuPAGE, BioRad) and stained with Coomassie Blue (BioSafe, BioRad). The arrows and asterisks indicate the protein monomers and aggregates, respectively.

Fig 4. Endemic and P. knowlesi-negative sera reactivity to Plasmodium knowlesi-specific antigens.

Scatter plots showing sera reactivity to: P. vivax MSP1-19 with P. knowlesi SERA3 ag1 (column 1), SERA3 ag2 (column 2), SSP2/TRAP (column 3) and TSERA2 ag1 (column 4) antigens. Sera samples from P. knowlesi-negative controls n = 55 (row 1; PHE UK malaria naïve (blue), Ethiopian children (red)) and Malaysian hospital case sera samples from days 0 (n = 92), 7 (n = 72) and 28 (n = 77) of diagnosis (rows 2–4, respectively). The red line in each graph represent the cut off values for the respective P. knowlesi antigen and was calculated based on Public Health England negative control sera samples (average ODs ± (3xSD)): The vertical cut off line is based on PvMSP1-19 = 0.501. The horizontal cut off line for the P. knowlesi antigens were based on the following values: SERA3 ag1 = 0.292; SERA3 ag2 = 0.366; SSP2/TRAP = 0.322 and TSERA2 ag1 = 0.208.

Fig 5. Serial fold increase in antibody reactivity for each antigen following treatment of knowlesi malaria.

(a) SERA3 ag1, (b) SERA3 ag2, (c) SSP2/TRAP and (d) TSERA2 ag1. Asterisks indicate level of significance, ns denotes non-significant values (p≤0.0001 ****; p≤0.001 ***; p≤0.01 **; p≤0.05 * and p>0.05 ns).

Fig 6. Relative variable importance of responses to each antigen from 100 boosted regression tree models predicting P. knowlesi seropositivity.

Median values for the relative variable importance and interquartile ranges are shown for all antigens tested: SERA3 ag 1 (4.8%; IQR 2.5–7.8%); SERA3 ag 2 (50.4%; IQR 43.3–61.4%); PkSSP2/TRAP (6.5%; IQR 3.7–11.8%) and TSERA ag 1 (34.2%; IQR 26.2–41.8%).