Symptomatic malaria enhances protection from reinfection with homologous Plasmodium falciparum parasites

Abstract

A signature remains elusive of naturally-acquired immunity against Plasmodium falciparum. We identified P. falciparum in a 14-month cohort of 239 people in Kenya, genotyped at immunogenic parasite targets expressed in the pre-erythrocytic (circumsporozoite protein, CSP) and blood (apical membrane antigen 1, AMA-1) stages, and classified into epitope type based on variants in the DV10, Th2R, and Th3R epitopes in CSP and the c1L region of AMA-1. Compared to asymptomatic index infections, symptomatic malaria was associated with reduced reinfection by parasites bearing homologous CSP-Th2R (adjusted hazard ratio [aHR]:0.63; 95% CI:0.45-0.89; p = 0.008) CSP-Th3R (aHR:0.71; 95% CI:0.52-0.97; p = 0.033), and AMA-1 c1L (aHR:0.63; 95% CI:0.43-0.94; p = 0.022) epitope types. The association of symptomatic malaria with reduced hazard of homologous reinfection was strongest for rare epitope types. Symptomatic malaria provides more durable protection against reinfection with parasites bearing homologous epitope types. The phenotype represents a legible molecular epidemiologic signature of naturally-acquired immunity by which to identify new antigen targets.

Fig 1. Calculation of time-to-reinfection based on infection episodes and on type-specific episodes.

Illustration using mock data of the calculation of time-to-reinfection for 3 participants with multiple episodes. Dots indicate beginning and end of episodes, which are joined by horizontal bars. Gray shaded area indicates the time period of continuous P. falciparum positivity. Labels between bars indicate either time-to-reinfection (in days) or censoring following the end of an episode. (A) Pf infection indicates episodes of infection with P. falciparum. (B) Epitope X indicate episodes of infection with specific epitope types of P. falciparum, which are classified on the basis of translated nucleotide sequences of csp and ama1. Colors indicate unique CSP or AMA-1 epitopes which comprise multi-clonal infections of P falciparum.

Fig 2. Risk of reinfection overall and as a function of symptomaticity of the index infection.

A) Kaplan-Meier survival curve of time to reinfection with P. falciparum. The black line is the probability among all participants, and colored lines are probability stratified by index case as either asymptomatic (Asx, blue) or symptomatic (Sx, red). Note that the blue line is largely hidden by the black line. Crosses indicate infections that were not followed by a reinfection and therefore censored at the end of the study period. Vertical dotted line indicates the start of the defined time at risk of re-infection, shaded areas indicate the 95% confidence interval, and table shows the number of participants at risk stratified by symptomaticity of index infection at day 0, 100, 200, 300, and 400. B) Adjusted Hazard Ratios for reinfection estimated by a multivariate mixed effects Cox proportional hazards model. See methods for variable definitions. Diamonds indicate adjusted HRs, and bars show the 95% CIs. Adj. HR: Adjusted Hazard Ratio.

Fig 3. Amino acid diversity of sequenced pfcsp and ama1 fragments.

A) Sequence logo of the observed circumsporozoite protein (CSP) amino acid variation. The size of the letters at each position shows the probability of the specific residue. Boxes indicate the positions of known epitopes for B-cells (DV10, blue), CD4⁺ T-cells (Th2R, red) and CD8⁺ T-cells (Th3R, yellow). B) Result of a random forest model used to predict the whole CSP fragment nucleotide sequence using amino acid variation. Bar height indicates for each variant amino acid position the mean decrease in accuracy (MDA) of prediction. Gold indicates the four most predictive amino acid positions within each T-cell epitope. C) Sequence logo of the observed apical membrane antigen 1 (AMA-1) amino acid variation. The size of the letters at each position shows the probability of the specific residue. Green box marks the c1L domain. D) The mean decrease in accuracy for each variant amino acid position in a random forest model used to predict the whole AMA-1 fragment nucleotide sequence. Gold indicates the four most predictive amino acid positions within the c1L domain.

Fig 4. Time to reinfection by homologous P. falciparum parasites stratified by symptomaticity of index infection.

A) Kaplan-Meier survival curves of time to reinfection by parasites with homologous CSP-Th2R, CSP-Th3R, and AMA-1 c1L type. Curves are probability stratified by index case as either asymptomatic (Asx, blue) or symptomatic (Sx, red). Crosses indicate index infections that were censored at the end of the study period. Vertical dotted line indicates the start of the defined time at risk of re-infection, shaded areas indicate the 95% confidence interval, and table shows the number of participants at risk stratified by symptomaticity of index infection at day 0, 100, 200, 300, and 400. B) Adjusted Hazard Ratios estimated by multivariate mixed effects Cox proportional hazards models for reinfection by parasites bearing homologous CSP-Th2R, CSP-Th3R, and AMA-1 c1L epitope types. See methods for variable definitions and reference cateogries. Diamonds indicate adjusted HRs, and bars show the 95% CIs. Adj. HR, Adjusted Hazard Ratio.

Fig 5. Prevalence of epitope types and time to homologous reinfection following asymptomatic and symptomatic infections.

Left: Gray bars indicate the number of infections harboring each CSP-Th2R (A), CSP-Th3R (B), or AMA-1 c1L (C) epitope type (right y-axis). Dots indicate the proportion of each index infection harboring the epitope type that was followed by homologous reinfection after 1 year (left y-axis) for either symptomatic (red) or asymptomatic (blue) index infections, with lines as standard error. Right: Adjusted Hazard Ratios for symptomatic index infections estimated by multivariate mixed effects Cox proportional hazards models for reinfection by parasites bearing homologous CSP-Th2R epitopes (A), CSP-Th3R (B), or AMA-1 c1L (C) epitopes that are individually classified as common, middling, and rare, as indicated in the bar plot. Hazard ratios adjusted for age, epitope type prevalence, gender, and transmission season. Circles indicate adjusted HRs, and bars show the 95% CIs. Adj. HR, Adjusted Hazard Ratio.