Natural antibody response to Plasmodium falciparum merozoite antigens MSP5, MSP9 and EBA175 is associated to clinical protection in the Brazilian Amazon

BACKGROUND: Antibodies have an essential role in the acquired immune response against blood stage P. falciparum infection. Although several antigens have been identified as important antibody targets, it is still elusive which antigens have to be recognized for clinical protection. Herein, we analyzed antibodies from plasmas from symptomatic or asymptomatic individuals living in the same geographic area in the Western Amazon, measuring their recognition of multiple merozoite antigens. METHODS: Specific fragments of genes encoding merozoite proteins AMA1 and members of MSP and EBL families from circulating P. falciparum field isolates present in asymptomatic and symptomatic patients were amplified by PCR. After cloning and expression of different versions of the antigens as recombinant GST-fusion peptides, we tested the reactivity of patients’ plasmas by ELISA and the presence of IgG subclasses in the most reactive plasmas. RESULTS: 11 out of 24 recombinant antigens were recognized by plasmas from either symptomatic or asymptomatic infections. Antibodies to MSP9 (X(2)(DF=1) = 9.26/p = 0.0047) and MSP5 (X(2)(DF=1) = 8.29/p = 0.0069) were more prevalent in asymptomatic individuals whereas the opposite was observed for MSP1 block 2-MAD20 (X(2)(DF=1) = 6.41/p = 0.0206, Fisher’s exact test). Plasmas from asymptomatic individuals reacted more intensely against MSP4 (U = 210.5, p < 0.03), MSP5 (U = 212, p < 0.004), MSP9 (U = 189.5, p < 0.002) and EBA175 (U = 197, p < 0.014, Mann-Whitney’s U test). IgG1 and IgG3 were predominant for all antigens, but some patients also presented with IgG2 and IgG4. The recognition of MSP5 (OR = 0.112, IC(95%) = 0.021-0.585) and MSP9 (OR = 0.125, IC(95%) = 0.030-0.529, cross tab analysis) predicted 8.9 and 8 times less chances, respectively, to present symptoms. Higher antibody levels against MSP5 and EBA175 were associated by odds ratios of 9.4 (IC(95%) = 1.29-69.25) and 5.7 (IC(95%) = 1.12-29.62, logistic regression), respectively, with an asymptomatic status. CONCLUSIONS: Merozoite antigens were targets of cytophilic antibodies and antibodies against MSP5, MSP9 and EBA175 were independently associated with decreased symptoms

<i>Plasmodium</i> dihydrofolate reductase is a second enzyme target for the antimalarial action of triclosan

Malaria, caused by parasites of the genus Plasmodium, leads to over half a million deaths per year, 90% of which are caused by Plasmodium falciparum. P. vivax usually causes milder forms of malaria; however, P. vivax can remain dormant in the livers of infected patients for weeks or years before re-emerging in a new bout of the disease. The only drugs available that target all stages of the parasite can lead to severe side effects in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency; hence, there is an urgent need to develop new drugs active against blood and liver stages of the parasite. Different groups have demonstrated that triclosan, a common antibacterial agent, targets the Plasmodium liver enzyme enoyl reductase. Here, we provide 4 independent lines of evidence demonstrating that triclosan specifically targets both wild-type and pyrimethamine-resistant P. falciparum and P. vivax dihydrofolate reductases, classic targets for the blood stage of the parasite. This makes triclosan an exciting candidate for further development as a dual specificity antimalarial, which could target both liver and blood stages of the parasite.This work was supported by: the UK Biotechnology and Biological Sciences Research Council (BB/F008228/1) and a contract from the European Commission under the FP7 Collaborative Programme, UNICELLSYS, both to S.G.O. and R.D.K.; the Bill and Melinda Gates foundation (Op1087646 to EB and SGO), São Paulo Research Foundation - FAPESP (2012/23306-5 to WLF, EFGC and GW and 2015/19103-0 and 2015/03553-6 to EB), the ERC (208813 to FH)

Dynamic Activation and Repression of the Plasmodium falciparum rif Gene Family and Their Relation to Chromatin Modification

The regulation of variant gene expression in Plasmodium falciparum is still only partially understood. Regulation of var genes, the most studied gene family involved in antigenic variation, is orchestrated by a dynamic pattern of inherited chromatin states. Although recent evidence pointed to epigenetic regulation of transcribed and repressed rif loci, little is known about specific on/off associated histone modifications of individual rif genes. To investigate the chromatin marks for transcribed and repressed rif loci, we cultivated parasites and evaluated the transcriptional status of chosen rif targets by qRT-PCR and performed ChIP assays using H3K9ac and H3K9me3 antibodies. We then monitored changes in the epigenetic patterns in parasites after several reinvasions and also evaluated the “poised” mark in trophozoites and schizonts of the same erythrocytic cycle by ChIP using H3K4me2 specific antibodies. Our results show that H3K9 is acetylated in transcribed rif loci and trimethylated or even unmodified in repressed rif loci. These transcriptional and epigenetic states are inherited after several reinvasions. The poised modification H3K4me2 showed a tendency to be more present in loci in trophozoites that upon progression to schizonts strongly transcribe the respective locus. However, this effect was not consistently observed for all monitored loci. While our data show important similarities to var transcription-associated chromatin modifications, the observed swiftly occurring modifications at rif loci and the absence of H3K9 modification point to a different dynamic of recruitment of chromatin modifying enzymes

<i>Rif</i> transcription and H3K9 modifications after reselection over CHO-CD36 cells.

<p>(A) The <i>rif</i> transcript quantities of freshly CHO-CD36 selected parasites in trophozoite stage (20–24 h post invasion) were monitored by RT-qPCR in relation to the internal control PF070073. (B) Histone modifications were measured by ChIP as described and significant differences in signals between H3K9ac and H3K9me3 modifications are depicted by asterisks (Student's T test, <i>p</i><0.05). ChIP was performed in triplicates.</p

Relative quantities of <i>rif</i> transcripts and chromatin enrichment for H3K9ac, H3K9me3 and H3K4me2 (poised mark) in trophozoite and schizont stages for selected <i>rif</i> loci.

<p>A: Relative transcript amounts of chosen <i>rif</i> targets in trophozoites and schizonts in 3D7 parasites of the same reinvasion, calculated as above. B: Ratios of H3K9ac/me3 enrichment in trophozoites (20–24 h p.i.) and schizonts (30–34 h p.i.). C: Scatter plots of the relative transcription in schizonts plotted against the H3K4me2 enrichment in trophozoites showing a positive and significant correlation (R² = 0.4929, p = 0.02). The ChIP experiment was done in biological triplicates.</p

Transcriptional analysis and chromatin enrichment after 10 and 20 reinvasions.

<p>A: Transcription of 3D7-CD36 parasites of chosen <i>rif</i> targets are shown as relative values (2^−ΔCt) using the gene of plasmodial t-seryl Synthetase (PF070073) as an internal control. B, C: Chromatin enrichment at selected <i>rif</i> loci shown as H3K9ac/H3K9me3 ratios obtained for 20–24 h trophozoite samples after 10 (B) and 20 reinvasions (C). D: Scatter plot showing the relation of histone modifications and transcript quantity. The coefficients of relative transcription at each loci (at 20 and 10 reinvasions) were plotted against the coefficient of H3K9ac/me3 ratios_{20 reinvasions}/H3K9ac/me3 ratios_{10 reinvasions}. The ChIP experiment was done in biological triplicates and Pearson's correlation of the values is indicated (p = 0.03, one tailed probability). Note that the extreme outliers PF080138 and PFB0040c were not considered.</p