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Neutrophils and macrophages confine pathogens by entrapping them in extracellular traps (ETs) through activating TLR9 function. However, plasmodial parasites secreted TatD-like DNases (TatD) to counteract ETs-mediated immune clearance. We found that TLR9 mutant mice increased susceptibility to rodent malaria, suggesting TLR9 is a key protein for host defense. We found that the proportion of neutrophils and macrophages in response to plasmodial parasite infection in the TLR9 mutant mice was significantly reduced compared to that of the WT mice. Importantly, PbTatD can directly bind to the surface TLR9 (sTLR9) on macrophages, which blocking the phosphorylation of mitogen-activated protein kinase and nuclear factor-κB, negatively regulated the signaling of ETs formation by both macrophages and neutrophils. Such, P. berghei TatD is a parasite virulence factor that can inhibit the proliferation of macrophages and neutrophils through directly binding to TLR9 receptors on the cell surface, thereby blocking the activation of the downstream MyD88-NF-kB pathways., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Artemisia annua is an anti-fever herbal medicine first described in traditional Chinese medicine 1,000 years ago. Artemisinin, the extract of A. annua, and its derivatives (dihydroartemisinin (DHA), artemether, and artesunate) have been used for the treatment of malaria with substantial efficacy. Recently, DHA has also been tested for the treatment of lupus erythematosus, indicating that it may function to balance the immune response in immunocompromised individuals. In the present study, the regulatory effect of artemisinin on the murine immune system was systematically investigated in mice infected with two different protozoan parasites (Toxoplasma gondii and Plasmodium berghei). Our results revealed that the mouse spleen index significantly increased (spleen enlargement) in the healthy mice after DHA administration primarily due to the generation of an extra number of lymphocytes and CD8 + T lymphocytes in both the spleen and circulation. DHA could increase the proportion of T helper cells and CD8 + T cells, as well as decrease the number of splenic and circulatory B cells. Further, DHA could reduce the production of proinflammatory cytokines. Our study revealed that apart from their anti-parasitic activity, artemisinin and its derivatives can also actively modulate the immune system to directly benefit the host.

Background: Malaria, one of the largest health burdens worldwide, is caused by Plasmodium spp. infection. Upon infection, the host's immune system begins to clear the parasites. However, Plasmodium species have evolved to escape the host's immune clearance. T-cell immunoglobulin and mucin domain 3 (Tim-3), a surface molecule on most immune cells, is often referred to as an exhaustion marker. Galectin (Gal)-9 is a Tim-3 ligand and the T helper (Th) 1 cell response is inhibited when Gal-9 binds to Tim-3. In the present study, dynamic expression of Tim-3 on key populations of lymphocytes during infection periods of Plasmodium berghei and its significance in disease resistance and pathogenesis were explored., Methods: Tim-3 expression on critical lymphocyte populations and the proportion of these cells, as well as the levels of cytokines in the sera of infected mice, were detected by flow cytometry. Further, in vitro anti-Tim-3 assay using an anti-Tim-3 antibody and in vivo Tim-3-Gal-9 signaling blockade assays using α-lactose (an antagonist of Gal-9) were conducted. An Annexin V Apoptosis Detection Kit with propidium iodide was used to detect apoptosis. In addition, proteins associated with apoptosis in lung and spleen tissues were confirmed by Western blotting assays., Results: Increased Tim-3 expression on splenic CD8 + and splenic CD4 + , and circulatory CD4 + T cells was associated with a reduction in the proportion of these cells. Furthermore, the levels of interleukin (IL)-2, IL-4, IL-6, IL-22, and interferon (IFN)-γ, but not that of tumor necrosis factor alpha (TNF-α), IL-10, and IL-9, increased to their highest levels at day 4 post-infection and decreased thereafter. Blocking Tim-3 signaling in vitro inhibited lymphocyte apoptosis. Tim-3-Gal-9 signaling blockade in vivo did not protect the mice, but induced the expression of the immunosuppressive molecule, T cell immunoreceptor with Ig and ITIM domains (TIGIT), in Plasmodium berghei ANKA-infected mice., Conclusions: Tim-3 on lymphocytes negatively regulates cell-mediated immunity against Plasmodium infection, and blocking Tim-3-galectin 9 signaling using α-lactose did not significantly protect the mice; however, it induced the compensatory expression of TIGIT. Further investigations are required to identify whether combined blockade of Tim-3 and TIGIT signaling could achieve a better protective effect.

Cell-mediated immune responses play important roles in immune protection against Plasmodium infection. However, impaired immunity, such as lymphocyte exhaustion, is a common phenomenon in malaria. T-cell immunoglobulin- and mucin-domain-containing molecule 3 (Tim-3) is an important regulatory molecule in cell-mediated immunity and has been implicated in malaria. In this study, it was found that Tim-3 expression on key populations of lymphocytes was significantly increased in both Plasmodium falciparum-infected patients and Plasmodium berghei ANKA (PbANKA)-infected C57BL/6 mice. Upregulation of Tim-3 led to lymphocyte exhaustion, while blocking Tim-3 signaling with an anti-Tim-3 antibody restored lymphocyte activity in Plasmodium infections. Further, anti-Tim-3 treatment accelerated the parasite clearance and relieved the symptoms of cerebral malaria in PbANKA-infected mice. In conclusion, Tim-3 on immune cells negatively regulates cell-mediated immunity against Plasmodium infection, and blocking Tim-3 signaling enhances sterile immunity and may play a protective role during malarial parasite infections., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

Background: The simian malaria parasite, Plasmodium knowlesi, can cause severe and fatal disease in humans yet it is rarely included in routine public health reporting systems for malaria and its geographical range is largely unknown. Because malaria caused by P. knowlesi is a truly neglected tropical disease, there are substantial obstacles to defining the geographical extent and risk of this disease. Information is required on the occurrence of human cases in different locations, on which non-human primates host this parasite and on which vectors are able to transmit it to humans. We undertook a systematic review and ranked the existing evidence, at a subnational spatial scale, to investigate the potential geographical range of the parasite reservoir capable of infecting humans., Methodology/principal Findings: After reviewing the published literature we identified potential host and vector species and ranked these based on how informative they are for the presence of an infectious parasite reservoir, based on current evidence. We collated spatial data on parasite occurrence and the ranges of the identified host and vector species. The ranked spatial data allowed us to assign an evidence score to 475 subnational areas in 19 countries and we present the results on a map of the Southeast and South Asia region., Conclusions/significance: We have ranked subnational areas within the potential disease range according to evidence for presence of a disease risk to humans, providing geographical evidence to support decisions on prevention, management and prophylaxis. This work also highlights the unknown risk status of large parts of the region. Within this unknown category, our map identifies which areas have most evidence for the potential to support an infectious reservoir and are therefore a priority for further investigation. Furthermore we identify geographical areas where further investigation of putative host and vector species would be highly informative for the region-wide assessment.

To determine the frequency of co-infections with Plasmodium species in southern Myanmar, we investigated the prevalence of P. knowlesi. More than 20% of patients with malaria had P. knowlesi infection, which occurred predominantly as a co-infection with either P. falciparum or P. vivax.

The sporozoites of Plasmodium parasites undergo several transmigrations before their establishment in the hepatocytes of a vertebrate host. Techniques that illustrate parasite intra-vital migration and their interaction with host cells will advance the understanding of parasite biology. In a recent publication, Amino et al. provided a detailed protocol for in vivo imaging of Plasmodium berghei sporozoites in the dermis. The report has important implications in the dissection of malaria parasite biology.

Although malaria and Epstein-Barr (EBV) infection are recognized cofactors in the genesis of endemic Burkitt lymphoma (BL), their relative contribution is not understood. BL, the most common paediatric cancer in equatorial Africa, is a high-grade B cell lymphoma characterized by c-myc translocation. EBV is a ubiquitous B lymphotropic virus that persists in a latent state after primary infection, and in Africa, most children have sero-converted by 3 y of age. Malaria infection profoundly affects the B cell compartment, inducing polyclonal activation and hyper-gammaglobulinemia. We recently identified the cystein-rich inter-domain region 1alpha (CIDR1alpha) of the Plasmodium falciparum membrane protein 1 as a polyclonal B cell activator that preferentially activates the memory compartment, where EBV is known to persist. Here, we have addressed the mechanisms of interaction between CIDR1alpha and EBV in the context of B cells. We show that CIDR1alpha binds to the EBV-positive B cell line Akata and increases the number of cells switching to the viral lytic cycle as measured by green fluorescent protein (GFP) expression driven by a lytic promoter. The virus production in CIDR1alpha-exposed cultures was directly proportional to the number of GFP-positive Akata cells (lytic EBV) and to the increased expression of the EBV lytic promoter BZLF1. Furthermore, CIDR1alpha stimulated the production of EBV in peripheral blood mononuclear cells derived from healthy donors and children with BL. Our results suggest that P. falciparum antigens such as CIDR1alpha can directly induce EBV reactivation during malaria infection that may increase the risk of BL development for children living in malaria-endemic areas. To our knowledge, this is the first report to show that a microbial protein can drive a latently infected B cell into EBV replication.

Chronic malaria infection is characterized by polyclonal B cell activation, hyperglobulinemia, and elevated titers of autoantibodies. We have recently identified the cysteine-rich interdomain region 1alpha (CIDR1alpha) of the Plasmodium falciparum erythrocyte membrane protein 1 as a T cell-independent polyclonal B cell activator and Ig binding protein. Here, we show that, although the binding affinity of CIDR1alpha to human IgM and IgG is relatively low, B cell activation still proceeds. CIDR1alpha rescues tonsillar B cells from apoptosis, and increases the proportion of cycling cells. Comparison of the impact on naive and memory B cell compartment indicated that CIDR1alpha preferentially activates memory B lymphocytes. Analysis of the gene expression profiles induced by CIDR1alpha and anti-Ig activation using a cDNA microarray demonstrated a low degree of homology in the signatures imposed by both stimuli. The microarray data correlate with the functional analysis demonstrating that CIDR1alpha activates various immunological pathways and protects B cells from apoptosis. Together, the results provide evidence for a role of malaria in preferentially activating the memory B cell compartment. The polyclonal B cell activation and augmented survival induced by CIDR1alpha is of relevance for understanding the mechanisms behind the increased risk of Burkitt's lymphoma in malaria endemic areas.

The development of malaria vaccines and medicines depends on the discovery of novel malaria protein targets, but the functions of more than 40% of P. falciparum genes remain unknown. Asexual parasites are the critical stage that leads to serious clinical symptoms and that can be modulated by malaria treatments and vaccines. To identify critical genes involved in the development of Plasmodium parasites within erythrocytes, the expression profile of more than 5,000 genes distributed across the 14 chromosomes of the PF3D7 strain during its six critical developmental stages (merozoite, early-ring, late-ring, early trophozoite, late-trophozoite, and middle-schizont) was evaluated. Hence, a qRT-PCR-based transcriptome of the erythrocytic developmental process of P. falciparum was revealed. Weighted gene coexpression network analyses revealed that a large number of genes are upregulated during the merozoite release process. Further gene ontology analysis revealed that a cluster of genes is associated with merozoite and may be apical complex components. Among these genes, 135 were comprised within chromosome 14, and 80% of them were previously unknown in functions. Western blot and immunofluorescence assays using newly developed corresponding antibodies showed that some of these newly discovered proteins are highly expressed in merozoites. Further invasion inhibition assays revealed that specific antibodies against several novel merozoite proteins can interfere with parasite invasion. Taken together, our study provides a developmental transcriptome of the asexual parasites of P. falciparum and identifies a group of previously unknown merozoite proteins that may play important roles in the process of merozoite invasion. [ABSTRACT FROM AUTHOR]

Plasmodium falciparum extensively remodels host cells by translocating numerous proteins into the cytoplasm of red blood cells (RBCs) after invasion. Among these exported proteins, members of the Plasmodium helical interspersed subtelomeric (PHIST) family are crucial for host cell remodeling and host-parasite interactions, and thereby contribute to malaria pathogenesis. Herein, we explored the function of PF3D7_1372300, a member of the PHIST/PHISTa-like subfamily. PF3D7_1372300 was highly transcribed and expressed during the blood stage of P. falciparum , and distributed throughout RBCs, but most abundant at the erythrocyte membrane. Specific interaction of PF3D7_1372300 with the cytoplasmic tail of P. falciparum erythrocyte membrane protein 1 (PfEMP1) was revealed by immunofluorescence assay, in vitro intermolecular interaction assays. The interaction sites of PF3D7_1372300 with PfEMP1 ATS domain were found involved more than 30 amino acids (aa) at several positions. The findings deepen our understanding of host-parasite interactions and malaria pathogenesis. [ABSTRACT FROM AUTHOR]

Mutually exclusive gene expression, whereby only one member of a multi-gene family is selected for activation, is used by the malaria parasite Plasmodium falciparum to escape the human immune system and perpetuate long-term, chronic infections. A family of genes called var encodes the chief antigenic and virulence determinant of P. falciparum malaria. var genes are transcribed in a mutually exclusive manner, with switching between active genes resulting in antigenic variation. While recent work has shed considerable light on the epigenetic basis for var gene activation and silencing, how switching is controlled remains a mystery. In particular, switching seems not to be random, but instead appears to be coordinated to result in timely activation of individual genes leading to sequential waves of antigenically distinct parasite populations. The molecular basis for this apparent coordination is unknown. Here we show that var2csa, an unusual and highly conserved var gene, occupies a unique position within the var gene switching hierarchy. Induction of switching through the destabilization of var specific chromatin using both genetic and chemical methods repeatedly led to the rapid and exclusive activation of var2csa. Additional experiments demonstrated that these represent “true” switching events and not simply de-silencing of the var2csa promoter, and that activation is limited to the unique locus on chromosome 12. Combined with translational repression of var2csa transcripts, frequent “default” switching to this locus and detection of var2csa untranslated transcripts in non-pregnant individuals, these data suggest that var2csa could play a central role in coordinating switching, fulfilling a prediction made by mathematical models derived from population switching patterns. These studies provide the first insights into the mechanisms by which var gene switching is coordinated as well as an example of how a pharmacological agent can disrupt antigenic variation in Plasmodium falciparum. [ABSTRACT FROM AUTHOR]

Background: The simian malaria parasite, Plasmodium knowlesi, can cause severe and fatal disease in humans yet it is rarely included in routine public health reporting systems for malaria and its geographical range is largely unknown. Because malaria caused by P. knowlesi is a truly neglected tropical disease, there are substantial obstacles to defining the geographical extent and risk of this disease. Information is required on the occurrence of human cases in different locations, on which non-human primates host this parasite and on which vectors are able to transmit it to humans. We undertook a systematic review and ranked the existing evidence, at a subnational spatial scale, to investigate the potential geographical range of the parasite reservoir capable of infecting humans. Methodology/Principal Findings: After reviewing the published literature we identified potential host and vector species and ranked these based on how informative they are for the presence of an infectious parasite reservoir, based on current evidence. We collated spatial data on parasite occurrence and the ranges of the identified host and vector species. The ranked spatial data allowed us to assign an evidence score to 475 subnational areas in 19 countries and we present the results on a map of the Southeast and South Asia region. Conclusions/Significance: We have ranked subnational areas within the potential disease range according to evidence for presence of a disease risk to humans, providing geographical evidence to support decisions on prevention, management and prophylaxis. This work also highlights the unknown risk status of large parts of the region. Within this unknown category, our map identifies which areas have most evidence for the potential to support an infectious reservoir and are therefore a priority for further investigation. Furthermore we identify geographical areas where further investigation of putative host and vector species would be highly informative for the region-wide assessment. [ABSTRACT FROM AUTHOR]

Abstract: Malaria vaccine development has so far been largely focused on antigens involved in parasite invasion pathways rather than on antigens associated with severe disease and naturally acquired immunity. Individuals repeatedly exposed to Plasmodium falciparum will eventually become immune to severe disease. Parasite-derived antigens expressed on the infected red blood cell (iRBC) surface are the main targets of protective immunity and can be explored as a rational alternative in development of an anti-malaria vaccine. [Copyright &y& Elsevier]

Severe human malaria is attributable to an excessive sequestration of Plasmodium falciparum-infected and uninfected erythrocytes in vital organs. Strains of P. falciparum that form rosettes and employ heparan sulfate as a host receptor are associated with development of severe forms of malaria. Heparin, which is similar to heparan sulfate in that it is composed of the same building blocks, was previously used in the treatment of severe malaria, but it was discontinued due to the occurrence of serious side effects such as intracranial bleedings. Here we report to have depolymerized heparin by periodate treatment to generate novel glycans (dGAG) that lack anticoagulant-activity. The dGAGs disrupt rosettes, inhibit merozoite invasion of erythrocytes and endothelial binding of P. falciparum-infected erythrocytes in vitro, and reduce sequestration in in vivo models of severe malaria. An intravenous injection of dGAGs blocks up to 80% of infected erythrocytes from binding in the micro-vasculature of the rat and releases already sequestered parasites into circulation. P. falciparum-infected human erythrocytes that sequester in the non-human primate Macaca fascicularis were similarly found to be released in to the circulation upon a single injection of 500 μg of dGAG. We suggest dGAGs to be promising candidates for adjunct therapy in severe malaria. [ABSTRACT FROM AUTHOR]

A family of parasite antigens known as Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is believed to play an important role in the binding of infected erythrocytes to host receptors in the micro-vasculature. Available data advocates the existence of a subset of very adhesive (rosetting, auto-agglutinating) and antigenic PfEMP1s implicated as virulence factors. Serum antibodies that disrupt rosettes are rarely found in children with severe malaria but are frequent in those with mild disease suggesting that they may be protective. Here we have developed a Semliki forest virus (SFV) vaccine construct with a recombinant gene (mini-var gene) encoding a mini-PfEMP1 (DBL1α-TM-ATS) obtained from a particularly antigenic and rosetting parasite (FCR3S1.2). The mini-PfEMP1 is presented to the host mimicking the location of the native molecule at the infected erythrocyte surface. Antibodies generated by a regimen of priming with SFV RNA particles and boosting with a recombinant protein recognize the infected erythrocyte surface (immuno-fluorescence/rosette-disruption) and prevent the sequestration of P. falciparum-infected erythrocytes in an in vivo model of severe malaria. The data prove the involvement of DBL1α in the adhesion of infected- and uninfected erythrocytes and the role of rosette-disruptive antibodies in preventing these cellular interactions. The work supports the use of DBL1α in a vaccine again severe malaria. [Copyright &y& Elsevier]

Plasmodium falciparum being the most lethal plasmodiae is still a major cause of the disease burden and mortality in malaria endemic areas. Due to the wide spread drug resistance in combination with poor socio-economic situation in the vast majority of the endemic countries, malaria is today a great global challenge. The scientific community is, however, progressing. The 23 Mb genome of P. falciparum has been decoded and publicly available. Data of transcriptional profiling at certain developmental stages have already been generated. More than 50% of P. falciparum genes are transcribed constitutively in all the developmental stages of parasite life cycle. Functional disruption of these genes might have implications for parasite growth and development. Available microarray data indicate that P. falciparum preferentially expresses rif and stevor gene families at gametocyte and sporozoite stages while var genes are predominantly expressed at the erythrocytic stage. Gene regulation mechanisms of the variant gene families in P. falciparum are still not understood though some regulatory elements have been proposed. The occurrence of severe malaria is determined by both parasite and human host factors. Sequestration and antigenic variation are two of the evasion mechanisms utilized by P. falciparum in order to escape the human host defences. Understanding the molecular mechanisms underlying these phenomena is of a major importance and interest in malaria research. Here, we summarize and highlight the recent progress in molecular aspects of severe malaria. [Copyright &y& Elsevier]

Presents research which showed that multiple var gene transcripts can be detected in cultures of the rosetting parasite FCR3S1.2. Plasmodium falciparum the cause of lethal malaria; Binding of P. falciparum mediated by erythrocyte-membrane-protein-1 (PfEMP-1); Expression and switching of var genes; Var transcripts in single erythrocytes; Single P. falciparum parasite simultaneously transcribing multiple var genes.

Background: Plasmodium falciparum is the most virulent parasite of the five Plasmodium species that cause human malaria, and biological analysis of the parasite is critical for the development of novel strategies for disease control. DNA endonucleases are important for maintaining the biological activity, gene stability of the parasite and interaction with host immune systems. In this study, ten sequences of DNA endonucleases were found in the genome of P. falciparum 3D7 clone, seven of them were predicted to contain an endonuclease/exonuclease/phosphatase (IPR005135) domain which plays an important role in DNA catalytic activity. The seven DNA endonucleases of P. falciparum were systematically investigated. Methods: Plasmodium falciparum 3D7 clone was cultured in human O+ RBCs, RNA was extracted at 8, 16, 24, 32, 40, and 48 h post invasion and real-time quantitative PCR was carried out to analyse the transcription of the seven DNA endonuclease genes in asexual stages. Immunofluorescence assay was carried out to confirm the location of the encoded proteins expressed in the erythrocytic stages. Finally, the catalytic activity of the DNA nucleases were tested. Results: Of the seven proteins analysed, two proteins were not soluble. Fragments derived from the rest five endonuclease sequences were successfully expressed as soluble proteins, and which were used to generate antisera for protein localization. The proteins were all located in the nucleus at ring and trophozoite stages. While at schizont stage, proteins encoded by PF3D7_1238600, PF3D7_0107200 and PF3D7_0319200 were in the punctuated forms in the parasite most likely around nuclei of the merozoites. But the proteins encoded by PF3D7_0305600 and PF3D7_1363500 were distributed around the infected erythrocyte membrane. The enzymatic activity of the recombinant GST-PF3D7_1238600 was very efficient without divalent iron, while the activity of the rest four enzymes was iron dependent. Further, divalent irons did not show any specific enhancement on the activity of GST-PF3D7_1238600, but the activity of GST-PF3D7_0107200, GST-PF3D7_1363500 and GST-PF3D7_0319200 were Cu2+ dependent. The activity of GST-PF3D7_0305600 was dependent on Mg2+ and Mn2+. Except GST-PF3D7_1363500, four of the GST tagged recombinant proteins hydrolysed the supercoiled circular plasmid DNA with or without divalent metal ions. The GST-PF3D7_1363500 protein only changed the supercoiled circular plasmid DNA into nicked plasmids, even with Cu2+. Conclusions: Fragments derived from five of the endonuclease sequences of P. falciparum 3D7 clone were successfully expressed. The proteins displayed diverse cell distribution, biochemical and enzymatic activities, which indicated that they carried different biological function in the development of the parasite in the erythrocytes. The DNA repair and DNA degradation capacity of the DNA endonucleases in the biology of the parasite remained further studied. [ABSTRACT FROM AUTHOR]

Abstract: Antigenic variation is a survival mechanism developed by the malaria parasite Plasmodium falciparum in order to allow for the establishment of a chronic infection. Here we have studied clonal differences in the transcriptomes of two isogenic P. falciparum clones (3D7S8.4 and 3D7AH1S2) of distinct adhesive and antigenic phenotypes employing a P. falciparum 70-mer oligonucleotide microarray. Fifteen transcripts were highly differentially expressed (greater than a 5-fold change) with five transcripts upregulated in 3D7AH1S2 compared to 3D7S8.4, and ten downregulated. Identified genes encode apical organellar (Gbph2, GBP-related antigen), cell cycle and DNA/RNA processing (SERA-5, RNA-methylase), cell-rescue, defense/virulence (RESA-2, RIFIN, PfEMP1) and hypothetical proteins (PFB0115w, PFI1445w, MAL13P1.121). A number of short and full-length var transcripts were differentially expressed between the clones but one full-length transcript was dominant in both rings and trophozoites (PFD0630c versus PFF0845c). Distinct members of two other variant gene families (phist-a and rif-like), scattered over the subtelomeric areas of the 14 chromosomes, were also found to be clonally and developmentally expressed. Three sibling-clones of 3D7AH1S2 (3D7AH1S1, -S3, -S4) were further studied for the expression of transcripts upregulated in 3D7AH1S2 compared to 3D7S8.4. Individual var and phist-a genes were found expressed in all of the clones while the expression of a rif-like gene and gbph2 varied in-between the clones. The present data provides evidence for complex transcriptional differences between closely related isogenic P. falciparum of distinct adhesive and antigenic characteristics. [Copyright &y& Elsevier]

Abstract: The interactions of Plasmodium falciparum infected erythrocytes parasitized red blood cells (pRBC) with endothelial receptors and erythrocytes are mediated by multiple Duffy-binding like (DBL) and cysteine-rich interdomain region (CIDR) domains harboured in the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). The success of a subunit vaccine based on PfEMP1 depends on its ability to elicit cross-reactive responses to a substantial number of PfEMP1 variants. We have here evaluated serological PfEMP1 cross-reactivity by immunizing rats with phylogenetically diverse recombinant NTS-DBL-1α/x fusion domains from the 3D7 genome parasite emulsified in Montanide ISA 720. Cross-reactivity was elicited to these diverse DBL-1α/x domains as measured by ELISA and by immunoblotting. Employing a novel in vivo model of human infected erythrocyte sequestration, immunized animals were challenged with the FCR3S1.2 clone and cross-protection in terms of reduction in lung sequestration amounting to approximately 50% was demonstrated. Our results suggest that immunization with phylogenetically distant DBL-1α/x variants, can elicit partial cross-protection to challenge with the parasites harbouring a distant variant. These observations have implications for the design of multi-component vaccines against P. falciparum malaria. [Copyright &y& Elsevier]