METHODS: In order to investigate the efficacy of pre-clinical vaccine candidates in P. knowlesi-infected human cases, this study describes an in vitro invasion inhibition assay, using a P. knowlesi strain adapted to in vitro growth in human erythrocytes, PkA1-H.1. Recombinant proteins of P. knowlesi Duffy binding protein alpha (PkDBPα) and apical membrane antigen 1 (PkAMA1) were produced in Escherichia coli system and rabbit antibodies were generated from immune animals.
RESULTS: PkDBPα and PkAMA1 recombinant proteins were expressed as insoluble and produced as a functional refolded form for this study. Antibodies against PkDBPα and PkAMA1 specifically recognized recombinant proteins and native parasite proteins in schizont-stage parasites on the merozoite organelles. Single and combination of anti-PkDBPα and anti-PkAMA1 antibodies elicited strong growth inhibitory effects on the parasite in concentration-dependent manner. Meanwhile, IgG prevalence of PkDBPα and PkAMA1 were observed in 13.0 and 46.7% in human clinical patients, respectively.
CONCLUSION: These data provide support for the validation of in vitro growth inhibition assay using antibodies of DBPα and AMA1 in human-adapted P. knowlesi parasite PkA1-H.1 strain.
RESULTS: In the erythrocyte-binding assay, binding level was determined by scoring the number of rosettes that were formed by erythrocytes surrounding transfected mammalian COS-7 cells which expressed PkDBPαII. The assay result revealed a significant difference in the binding level. The number of rosettes scored for Fya+/b+ was 1.64-fold higher than that of Fya+/b- (155.50 ± 34.32 and 94.75 ± 23.16 rosettes, respectively; t(6) = -2.935, P = 0.026).
CONCLUSIONS: The erythrocyte-binding assay provided a simple approach to quantitatively determine the binding level of PkDBPαII to the erythrocyte Duffy antigen. Using this assay, PkDBPαII was found to display higher binding to Fya+/b+ erythrocytes than to Fya+/b- erythrocytes.
METHODS: We did an environmentally stratified, population-based, cross-sectional survey across households in the Kudat, Kota Marudu, Pitas, and Ranau districts in northern Sabah, Malaysia, encompassing a range of ecologies. Using blood samples, the transmission intensity of P knowlesi and other malaria species was measured by specific antibody prevalence and infection detected using molecular methods. Proportions and configurations of land types were extracted from maps derived from satellite images; a data-mining approach was used to select variables. A Bayesian hierarchical model for P knowlesi seropositivity was developed, incorporating questionnaire data about individual and household-level risk factors with selected landscape factors.
FINDINGS: Between Sept 17, 2015, and Dec 12, 2015, 10 100 individuals with a median age of 25 years (range 3 months to 105 years) were sampled from 2849 households in 180 villages. 5·1% (95% CI 4·8-5·4) were seropositive for P knowlesi, and marked historical decreases were observed in the transmission of Plasmodium falciparum and Plasmodium vivax. Nine Plasmodium spp infections were detected. Age, male sex, contact with macaques, forest use, and raised house construction were positively associated with P knowlesi exposure, whereas residing at higher geographical elevations and use of insecticide were protective. Agricultural and forest variables, such as proportions and fragmentation of land cover types, predicted exposure at different spatial scales from households.
INTERPRETATION: Although few infections were detected, P knowlesi exposure was observed in all demographic groups and was associated with occupational factors. Results suggest that agricultural expansion and forest fragmentation affect P knowlesi exposure, supporting linkages between land use change and P knowlesi transmission.
FUNDING: UK Medical Research Council, Natural Environment Research Council, Economic and Social Research Council, and Biotechnology and Biosciences Research Council.
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.
METHODOLOGY/PRINCIPAL FINDINGS: We conducted comprehensive surveys in three areas where P. knowlesi transmission is reported: Limbuak, Pulau Banggi and Matunggung, Kudat, Sabah, Malaysia and Bacungan, Palawan, the Philippines. Infection prevalence was low with parasites detected by PCR in only 0.2% (4/2503) of the population. P. knowlesi PkSERA3 ag1 antibody responses were detected in 7.1% (95% CI: 6.2-8.2%) of the population, compared with 16.1% (14.6-17.7%) and 12.6% (11.2-14.1%) for P. falciparum and P. vivax. Sero-prevalence was low in individuals <10 years old for P. falciparum and P. vivax consistent with decreased transmission of non-zoonotic malaria species. Results indicated marked heterogeneity in transmission intensity between sites and P. knowlesi exposure was associated with agricultural work (OR 1.63; 95% CI 1.07-2.48) and higher levels of forest cover (OR 2.40; 95% CI 1.29-4.46) and clearing (OR 2.14; 95% CI 1.35-3.40) around houses. Spatial patterns of P. knowlesi exposure differed from exposure to non-zoonotic malaria and P. knowlesi exposed individuals were younger on average than individuals exposed to non-zoonotic malaria.
CONCLUSIONS/SIGNIFICANCE: This is the first study to describe serological exposure to P. knowlesi and associated risk factors within endemic communities. Results indicate community-level patterns of infection and exposure differ markedly from demographics of reported cases, with higher levels of exposure among women and children. Further work is needed to understand these variations in risk across a wider population and spatial scale.
METHODS: Using the recently completed genome sequences from P. malariae, P. ovale and P. knowlesi, a set of 33 candidate cell surface and secreted blood-stage antigens was selected and expressed in a recombinant form using a mammalian expression system. These proteins were added to an existing panel of antigens from P. falciparum and P. vivax and the immunoreactivity of IgG, IgM and IgA immunoglobulins from individuals diagnosed with infections to each of the five different Plasmodium species was evaluated by ELISA. Logistic regression modelling was used to quantify the ability of the responses to determine prior exposure to the different Plasmodium species.
RESULTS: Using sera from European travellers with diagnosed Plasmodium infections, antigens showing species-specific immunoreactivity were identified to select a panel of 22 proteins from five Plasmodium species for serological profiling. The immunoreactivity to the antigens in the panel of sera taken from travellers and individuals living in malaria-endemic regions with diagnosed infections showed moderate power to predict infections by each species, including P. ovale, P. malariae and P. knowlesi. Using a larger set of patient samples and logistic regression modelling it was shown that exposure to P. knowlesi could be accurately detected (AUC = 91%) using an antigen panel consisting of the P. knowlesi orthologues of MSP10, P12 and P38.
CONCLUSIONS: Using the recent availability of genome sequences to all human-infective Plasmodium spp. parasites and a method of expressing Plasmodium proteins in a secreted functional form, an antigen panel has been compiled that will be useful to determine exposure to these parasites.