Rapid identification of genes controlling virulence and immunity in malaria parasites

Identifying the genetic determinants of phenotypes that impact disease severity is of fundamental importance for the design of new interventions against malaria. Here we present a rapid genome-wide approach capable of identifying multiple genetic drivers of medically relevant phenotypes within malaria parasites via a single experiment at single gene or allele resolution. In a proof of principle study, we found that a previously undescribed single nucleotide polymorphism in the binding domain of the erythrocyte binding like protein (EBL) conferred a dramatic change in red blood cell invasion in mutant rodent malaria parasites Plasmodium yoelii. In the same experiment, we implicated merozoite surface protein 1 (MSP1) and other polymorphic proteins, as the major targets of strain-specific immunity. Using allelic replacement, we provide functional validation of the substitution in the EBL gene controlling the growth rate in the blood stages of the parasites.This work was supported by the JSPS (project numbers Nos. JP25870525, JP24255009 and JP16K21233) (to RCu), A Royal Society Bilateral Grant for Co-operative Research (to RCa and RCu) and a Sasakawa Foundation Butterfield Award (to RCu), faculty baseline fund (BAS/1/1020-01-01) from the King Abdullah University of Science and Technology (KAUST) to AP, and Grants-in-Aid for Scientific Research on Innovative Areas JR23117008 (to OK). CJRI was supported by a Sir Henry Dale Fellowship, jointly funded by the Wellcome Trust and the Royal Society (101239/Z/13/Z)

The impact of preparedness in defying COVID-19 pandemic expectations in the Lowe Mekong Region: A case study

Dire COVID-19 expectations in the Lower Mekong Region (LMR) can be understood as Cambodia, the Lao PDR, Myanmar, Thailand, and Vietnam have stared down a succession of emerging infectious disease (EID) threats from neighboring China. Predictions that the LMR would be overwhelmed by a coming COVID-19 tsunami were felt well before the spread of the COVID-19 pandemic had been declared. And yet, the LMR, excepting Myanmar, has proved surprisingly resilient in keeping COVID-19 contained to mostly sporadic cases. Cumulative case rates (per one million population) for the LMR, including or excluding Myanmar, from January 1 to October 31 2020, are 1,184 and 237, respectively. More telling are the cumulative rates of COVID-19–attributable deaths for the same period of time, 28 per million with and six without Myanmar. Graphics demonstrate a flattening of pandemic curves in the LMR, minus Myanmar, after managing temporally and spatially isolated spikes in case counts, with negligible follow-on community spread. The comparable success of the LMR in averting pandemic disaster can likely be attributed to years of preparedness investments, triggered by avian influenza A (H5N1). Capacity building initiatives applied to COVID-19 containment included virological (influenza-driven) surveillance, laboratory diagnostics, field epidemiology training, and vaccine preparation. The notable achievement of the LMR in averting COVID-19 disaster through to October 31, 2020 can likely be credited to these preparedness measures

Low seroprevalence of COVID-19 in Lao PDR, late 2020

Background In 2020 Lao PDR had low reported COVID-19 cases but it was unclear whether this masked silent transmission. A seroprevalence study was done August - September 2020 to determine SARS-CoV-2 exposure. Methods Participants were from the general community (n=2433) or healthcare workers (n=666) in five provinces and bat/wildlife contacts (n=74) were from Vientiane province. ELISAs detected anti- SARS-CoV-2 Nucleoprotein (N; n=3173 tested) and Spike (S; n=1417 tested) antibodies. Double-positive samples were checked by IgM/IgG rapid tests. Controls were confirmed COVID-19 cases (n=15) and pre-COVID-19 samples (n=265). Seroprevalence for the general community was weighted to account for complex survey sample design, age and sex. Findings In pre-COVID-19 samples, 5·3%, [95% CI=3·1-8·7%] were anti-N antibody single-positive and 1·1% [0·3-3·5%] were anti-S antibody single positive. None were double positive. Anti-N and anti-S antibodies were detected in 5·2% [4·2-6·5%] and 2·1% [1·1-3·9%] of the general community, 2·0% [1·1-3·3%] and 1·4% [0·5-3·7%] of healthcare workers and 20·3% [12·6-31·0%] and 6·8% [2·8-15·3%] of bat/wildlife contacts. 0·1% [0·02-0·3%] were double positive for anti-N and anti-S antibodies (rapid test negative). Interpretation We find no evidence for significant SARS-CoV-2 circulation in Lao PDR before September 2020. This likely results from early decisive measures taken by the government, social behavior, and low population density. High anti-N /low anti-S seroprevalence in bat/wildlife contacts may indicate exposure to cross-reactive animal coronaviruses with threat of emerging novel viruses. Funding Agence Française de Développement. Additional; Institut Pasteur du Laos, Institute Pasteur, Paris and Luxembourg Ministry of Foreign and European Affairs (“PaReCIDS II”)

Site directed mutagenesis of <i>pyebl</i> AA position 351 reverses the phenotypes of parasites with slow and intermediate growth rates.

<p><b>(A)</b> Growth rate of <i>P. yoelii</i> strains 17X1.1pp, CU and of the CU-strains transfected with either CU (CU-EBL-351C>C) or 17X1.1 (CU-EBL-351C>Y) <i>Pyebl</i> gene in CBA mice inoculated with 1x106 iRBCs on Day 0. <b>(B)</b> Growth rate of <i>P. yoelii</i> strains 17X1.1pp, CU and of the 17X1.1pp-strains transfected with either 17X1.1 (17X1.1pp-EBL-351Y>Y) or CU (17X1.1pp-EBL-351Y>C) <i>Pyebl</i> gene alleles in CBA mice inoculated with 1x106 iRBCs on Day 0. Transfection with the 17X1.1pp (EBL-351Y) allele produces a significantly increased growth rate in the CU strain (CU-EBL-351C>C vs CU-EBL-351C>Y: p <0.01, Two-way ANOVA with Tukey post-test correction) that is not significantly different from 17X1.1pp growth rate following transfection with its native allele (17X1.1pp-EBL-351Y>Y vs. CU-EBL-351C>Y: p >0.05, Two-way ANOVA with Tukey post-test correction). Conversely, transfection with the CU (EBA-351C) allele significantly reduces growth (17X1.1pp-EBL-351Y>Y vs 17X1.1pp-EBL-351Y>C: p <0.01, Two-way ANOVA with Tukey post-test correction) and produces a phenotype that is not significantly different from CU transfected with its own allele (CU EBL-351C>C vs 17X1.1pp-EBL-351Y>C: p >0.05, Two-way ANOVA with Tukey post-test correction).</p

Genome-wide sequencing data.

<p><b>(A)</b> Genome-wide <i>Plasmodium yoelii</i> CU allele frequency of two independent genetic crosses grown in (a,b) naïve mice, (c,d) 17X1.1pp immunized mice and (e,f) CU-immunized mice. Light gray dots represent observed allele frequencies. Dark gray dots represent allele frequencies retained after filtering. Dark blue lines represent a smoothed approximation of the underlying allele frequency; a region of uncertainty in this frequency, of size three standard deviations, is shown in light blue. A conservative confidence interval describing the position of an allele evolving under selection is shown via a red bar. Allele frequencies are shown in log scale. <b>(B)</b> Evolutionary models fitted to allele frequency data. Filtered allele frequencies are shown as gray dots, while the model fit is shown as a red line. Dark blue and light blue vertical bars show combined and conservative confidence intervals for the location of the selected allele as reported in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006447#ppat.1006447.t003" target="_blank">Table 3</a>. Numbers in parentheses equate figures with locations in <b>(A)</b>. A black vertical line shows the position of a gene of interest.</p

Confidence intervals for driver locations as determined by mathematical modeling.

<p>Confidence intervals for driver locations as determined by mathematical modeling.</p

Localization of EBL.

<p>The C351Y polymorphism does not affect EBL subcellular localization in <i>Plasmodium yoelii</i>. <b>(A)</b> <i>P. yoelii</i> schizonts of wild type and transgenic parasite lines were incubated with fluorescent mouse anti-EBL serum, fluorescent rabbit anti-AMA1 serum, and DAPI nuclear staining. Colors indicate the localization of the <i>Pyebl</i>(green) and AMA-1 (red) proteins, as well as nuclear DNA (blue). 17XL: fast growing 17X clone previously shown to traffic EBL to the dense granules, not the micronemes, 17X1.1pp: 17x1.1pp strain, CU: CU strain, 17X1.1-351Y C: 17X1.1pp strain transfected with the CU allele for <i>Pyebl</i>, CU-351C Y: CU strain transfected with the 17X1.1pp allele of <i>Pyebl</i>. <b>(B)</b> The distance of EBL from AMA1 measured for five parasite strains and for 5–9 schizonts per strain; stars indicate p<0.01 using a Mann-Whitney U test. This indicates a shift in the location of <i>Pyebl</i> occurring in 17XL, but not in any other parasite lines.</p

EBL Amino acid sequence alignment of various malaria species and <i>Plasmodium yoelii</i> strains, and predicted protein structure consequences of the C351Y polymorphism.

<p><b>(A)</b> EBL orthologous and paralogous sequences from a variety of malaria species and <i>P. yoelii</i> strains were aligned using ClustalW. Only the amino acids surrounding position 351 are shown. The cysteine in positon 351 in <i>P. yoelii</i> is highly conserved across strains and species, with only strain 17X1.1pp bearing a C to Y substitution. PchAS: <i>Plasmodium chabaudi</i> AS strain; PbANKA: <i>Plasmodium berghei</i> ANKA strain; Py17X/17X1.1pp/CU/YM: <i>P. yoelii</i> 17X,17X1.1pp,CU,YM strains; Pk-DBLα/β/γ: <i>Plasmodium knowlesi</i> Duffy Binding Ligand alpha/beta/gamma (H strain); PvDBP: <i>Plasmodium vivax</i> Duffy Binding Protein (Sal-I strain);PcynB_DBP1/2: <i>Plasmodium cynomolgi</i> Duffy Binding Proteins 1/2 (B strain); Pf3D7_EBA140/175/181: <i>Plasmodium falciparum</i> Erythrocyte Binding Antigens 140/175/181 (3D7 strain). <b>(B)</b> Energy minimized homology model of the wild type <i>P. yoelii</i> (Py17XWT) Erythrocyte Binding Ligand (EBL). Inset depicts the disulfide bond between C351 and C420. (The protein is represented in cyan and the disulfide bonds are in yellow). <b>(C)</b> Energy minimized homology model of the mutant (C351Y) <i>P. yoelii</i> (Py17X1.1pp) Erythrocyte Binding Ligand (EBL). Inset depicts the lack of a disulfide bond between Y351 (substituted C351) and C420. (The protein is represented in cyan and the disulfide bonds are in yellow and Tyr351 [mutated] is represented in magenta).</p

Sudden changes in allele frequency identified using a jump-diffusion model.

<p>Details are given for loci at which a sudden jump in frequency was inferred with probability at least 1%. The latter value is the inferred probability that the change in allele frequency at a given locus arose from a jump to a random position between 0 and 1, as opposed to arising from a small change to the frequency at the previous locus. Data are shown for the naïve and 17-X immunized experiments; no jumps of this significance were inferred for the CU-immunized experiment.</p