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Controlled human malaria infections (CHMI) are a valuable tool to study parasite gene expression in vivo under defined conditions. In previous studies, virulence gene expression was analyzed in samples from volunteers infected with the Plasmodium falciparum (Pf) NF54 isolate, which is of African origin. Here, we provide an in-depth investigation of parasite virulence gene expression in malaria-naïve European volunteers undergoing CHMI with the genetically distinct Pf 7G8 clone, originating in Brazil. Differential expression of var genes, encoding major virulence factors of Pf, PfEMP1s, was assessed in ex vivo parasite samples as well as in parasites from the in vitro cell bank culture that was used to generate the sporozoites (SPZ) for CHMI (Sanaria PfSPZ Challenge (7G8)). We report broad activation of mainly B-type subtelomeric located var genes at the onset of a 7G8 blood stage infection in naïve volunteers, mirroring the NF54 expression study and suggesting that the expression of virulence-associated genes is generally reset during transmission from the mosquito to the human host. However, in 7G8 parasites, we additionally detected a continuously expressed single C-type variant, Pf7G8_040025600, that was most highly expressed in both pre-mosquito cell bank and volunteer samples, suggesting that 7G8, unlike NF54, maintains expression of some previously expressed var variants during transmission. This suggests that in a new host, the parasite may preferentially express the variants that previously allowed successful infection and transmission. Trial registration: ClinicalTrials.gov - NCT02704533; 2018-004523-36., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright: © 2023 Wichers-Misterek et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Malaria remains a major public health problem in many countries. Unlike influenza and HIV, where diversity in immunodominant surface antigens is understood geographically to inform disease surveillance, relatively little is known about the global population structure of PfEMP1, the major variant surface antigen of the malaria parasite Plasmodium falciparum. The complexity of the var multigene family that encodes PfEMP1 and that diversifies by recombination, has so far precluded its use in malaria surveillance. Recent studies have demonstrated that cost-effective deep sequencing of the region of var genes encoding the PfEMP1 DBLα domain and subsequent classification of within host sequences at 96% identity to define unique DBLα types, can reveal structure and strain dynamics within countries. However, to date there has not been a comprehensive comparison of these DBLα types between countries. By leveraging a bioinformatic approach (jumping hidden Markov model) designed specifically for the analysis of recombination within var genes and applying it to a dataset of DBLα types from 10 countries, we are able to describe population structure of DBLα types at the global scale. The sensitivity of the approach allows for the comparison of the global dataset to ape samples of Plasmodium Laverania species. Our analyses show that the evolution of the parasite population emerging out of Africa underlies current patterns of DBLα type diversity. Most importantly, we can distinguish geographic population structure within Africa between Gabon and Ghana in West Africa and Uganda in East Africa. Our evolutionary findings have translational implications in the context of globalization. Firstly, DBLα type diversity can provide a simple diagnostic framework for geographic surveillance of the rapidly evolving transmission dynamics of P. falciparum. It can also inform efforts to understand the presence or absence of global, regional and local population immunity to major surface antigen variants. Additionally, we identify a number of highly conserved DBLα types that are present globally that may be of biological significance and warrant further characterization., Competing Interests: The authors have declared that no competing interests exist.

The pathogenesis of Plasmodium falciparum malaria is linked to the variant surface antigen PfEMP1, which mediates tethering of infected erythrocytes to the host endothelium and is encoded by approximately 60 var genes per parasite genome. Repeated episodes of malaria infection result in the gradual acquisition of protective antibodies against PfEMP1 variants. The antibody repertoire is believed to provide a selective pressure driving the clonal expansion of parasites expressing unrecognized PfEMP1 variants, however, due to the lack of experimental in vivo models there is only limited experimental evidence in support of this concept. To get insight into the impact of naturally acquired immunity on the expressed var gene repertoire early during infection we performed controlled human malaria infections of 20 adult African volunteers with life-long malaria exposure using aseptic, purified, cryopreserved P. falciparum sporozoites (Sanaria PfSPZ Challenge) and correlated serological data with var gene expression patterns from ex vivo parasites. Among the 10 African volunteers who developed patent infections, individuals with low antibody levels showed a steep rise in parasitemia accompanied by broad activation of multiple, predominantly subtelomeric var genes, similar to what we previously observed in naïve volunteers. In contrast, individuals with intermediate antibody levels developed asymptomatic infections and the ex vivo parasite populations expressed only few var gene variants, indicative of clonal selection. Importantly, in contrast to parasites from naïve volunteers, expression of var genes coding for endothelial protein C receptor (EPCR)-binding PfEMP1 that are associated with severe childhood malaria was rarely detected in semi-immune adult African volunteers. Moreover, we followed var gene expression for up to six parasite replication cycles and demonstrated for the first time in vivo a shift in the dominant var gene variant. In conclusion, our data suggest that P. falciparum activates multiple subtelomeric var genes at the onset of blood stage infection facilitating rapid expansion of parasite clones which express PfEMP1 variants unrecognized by the host's immune system, thus promoting overall parasite survival in the face of host immunity., Competing Interests: BKLS (Executive Vice President Process Development and Manufacturing) and SLH (Chief Executive and Scientific Officer) are employees of Sanaria Inc. This affiliation does not alter our adherence to all PLOS policies on sharing data and materials. All other authors declare that no competing interests exist.

Within the human host, the malaria parasite Plasmodium falciparum is exposed to multiple selection pressures. The host environment changes dramatically in severe malaria, but the extent to which the parasite responds to-or is selected by-this environment remains unclear. From previous studies, the parasites that cause severe malaria appear to increase expression of a restricted but poorly defined subset of the PfEMP1 variant, surface antigens. PfEMP1s are major targets of protective immunity. Here, we used RNA sequencing (RNAseq) to analyse gene expression in 44 parasite isolates that caused severe and uncomplicated malaria in Papuan patients. The transcriptomes of 19 parasite isolates associated with severe malaria indicated that these parasites had decreased glycolysis without activation of compensatory pathways; altered chromatin structure and probably transcriptional regulation through decreased histone methylation; reduced surface expression of PfEMP1; and down-regulated expression of multiple chaperone proteins. Our RNAseq also identified novel associations between disease severity and PfEMP1 transcripts, domains, and smaller sequence segments and also confirmed all previously reported associations between expressed PfEMP1 sequences and severe disease. These findings will inform efforts to identify vaccine targets for severe malaria and also indicate how parasites adapt to-or are selected by-the host environment in severe malaria.

Virulence of the most deadly malaria parasite Plasmodium falciparum is linked to the variant surface antigen PfEMP1, which is encoded by about 60 var genes per parasite genome. Although the expression of particular variants has been associated with different clinical outcomes, little is known about var gene expression at the onset of infection. By analyzing controlled human malaria infections via quantitative real-time PCR, we show that parasite populations from 18 volunteers expressed virtually identical transcript patterns that were dominated by the subtelomeric var gene group B and, to a lesser extent, group A. Furthermore, major changes in composition and frequency of var gene transcripts were detected between the parental parasite culture that was used to infect mosquitoes and Plasmodia recovered from infected volunteers, suggesting that P. falciparum resets its var gene expression during mosquito passage and starts with the broad expression of a specific subset of var genes when entering the human blood phase.

Avoidance of antibody-mediated immune recognition allows parasites to establish chronic infections and enhances opportunities for transmission. The human malaria parasite Plasmodium falciparum possesses a number of multi-copy gene families, including var, rif, stevor and pfmc-2tm, which encode variant antigens believed to be expressed on the surfaces of infected erythrocytes. However, most studies of these antigens are based on in vitro analyses of culture-adapted isolates, most commonly the laboratory strain 3D7, and thus may not be representative of the unique challenges encountered by P. falciparum in the human host. To investigate the expression of the var, rif-A, rif-B, stevor and pfmc-2tm family genes under conditions that mimic more closely the natural course of infection, ex vivo clinical P. falciparum isolates were analyzed using a novel quantitative real-time PCR approach. Expression patterns in the clinical isolates at various time points during the first intraerythrocytic developmental cycle in vitro were compared to those of strain 3D7. In the clinical isolates, in contrast to strain 3D7, there was a peak of expression of the multi-copy gene families rif-A, stevor and pfmc-2tm at the young ring stage, in addition to the already known expression peak in trophozoites. Furthermore, most of the variant surface antigen families were overexpressed in the clinical isolates relative to 3D7, with the exception of the pfmc-2tm family, expression of which was higher in 3D7 parasites. Immunofluorescence analyses performed in parallel revealed two stage-dependent localization patterns of RIFIN, STEVOR and PfMC-2TM. Proteins were exported into the infected erythrocyte at the young trophozoite stage, whereas they remained inside the parasite membrane during schizont stage and were subsequently observed in different compartments in the merozoite. These results reveal a complex pattern of expression of P. falciparum multi-copy gene families during clinical progression and are suggestive of diverse functional roles of the respective proteins.

Plasmodium falciparum employs antigenic variation to evade the human immune response by switching the expression of different variant surface antigens encoded by the var gene family. Epigenetic mechanisms including histone modifications and sub-nuclear compartmentalization contribute to transcriptional regulation in the malaria parasite, in particular to control antigenic variation. Another mechanism of epigenetic control is the exchange of canonical histones with alternative variants to generate functionally specialized chromatin domains. Here we demonstrate that the alternative histone PfH2A.Z is associated with the epigenetic regulation of var genes. In many eukaryotic organisms the histone variant H2A.Z mediates an open chromatin structure at promoters and facilitates diverse levels of regulation, including transcriptional activation. Throughout the asexual, intraerythrocytic lifecycle of P. falciparum we found that the P. falciparum ortholog of H2A.Z (PfH2A.Z) colocalizes with histone modifications that are characteristic of transcriptionally-permissive euchromatin, but not with markers of heterochromatin. Consistent with this finding, antibodies to PfH2A.Z co-precipitate the permissive modification H3K4me3. By chromatin-immunoprecipitation we show that PfH2A.Z is enriched in nucleosomes around the transcription start site (TSS) in both transcriptionally active and silent stage-specific genes. In var genes, however, PfH2A.Z is enriched at the TSS only during active transcription in ring stage parasites. Thus, in contrast to other genes, temporal var gene regulation involves histone variant exchange at promoter nucleosomes. Sir2 histone deacetylases are important for var gene silencing and their yeast ortholog antagonises H2A.Z function in subtelomeric yeast genes. In immature P. falciparum parasites lacking Sir2A or Sir2B high var transcription levels correlate with enrichment of PfH2A.Z at the TSS. As Sir2A knock out parasites mature the var genes are silenced, but PfH2A.Z remains enriched at the TSS of var genes; in contrast, PfH2A.Z is lost from the TSS of de-repressed var genes in mature Sir2B knock out parasites. This result indicates that PfH2A.Z occupancy at the active var promoter is antagonized by PfSir2A during the intraerythrocytic life cycle. We conclude that PfH2A.Z contributes to the nucleosome architecture at promoters and is regulated dynamically in active var genes.

Cytoadherance of Plasmodium falciparum-infected erythrocytes in the brain, organs and peripheral microvasculature is linked to morbidity and mortality associated with severe malaria. Parasite-derived P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) molecules displayed on the erythrocyte surface are responsible for cytoadherance and undergo antigenic variation in the course of an infection. Antigenic variation of PfEMP1 is achieved by in situ switching and mutually exclusive transcription of the var gene family, a process that is controlled by epigenetic mechanisms. Here we report characterisation of the P. falciparum silent information regulator's A and B (PfSir2A and PfSir2B) and their involvement in mutual exclusion and silencing of the var gene repertoire. Analysis of P. falciparum parasites lacking either PfSir2A or PfSir2B shows that these NAD(+)-dependent histone deacetylases are required for silencing of different var gene subsets classified by their conserved promoter type. We also demonstrate that in the absence of either of these molecules mutually exclusive expression of var genes breaks down. We show that var gene silencing originates within the promoter and PfSir2 paralogues are involved in cis spreading of silenced chromatin into adjacent regions. Furthermore, parasites lacking PfSir2A but not PfSir2B have considerably longer telomeric repeats, demonstrating a role for this molecule in telomeric end protection. This work highlights the pivotal but distinct role for both PfSir2 paralogues in epigenetic silencing of P. falciparum virulence genes and the control of pathogenicity of malaria infection., Competing Interests: Competing interests. The authors have declared that no competing interests exist.