The Mats Wahlgren Group

Biology of Plasmodium falciparum

500,000 individuals are infected with P. falciparum malaria each day. Despite a 50% decrease of incidence and prevalence over the years, new approaches to eradicate the disease are still urgently needed. The emergence of drug resistant parasites and their ability to evade the host immune system, which prohibits the development of vaccines inducing sterile immunity, are constant re-occurring obstacles to current efforts in malaria endemic countries. Thus, the lack of success in eradicating malaria is partly due to the parasite’s remarkable ability to adapt to metabolic, immunological and pharmaceutical changes within the human host. Although we know that a high level of genome plasticity and a complex regulatory machinery of gene expression underlie this ability to adapt, the exact mechanisms used by the parasite to circumvent selective pressures are not understood. We hypothesize that by decoding the molecular details of the mechanisms that regulate genetic and non-genetic adaptation of P. falciparum to its host we will increase the understanding role of transcription, translation and switching of genes. This will enable the development of novel drugs that circumvent the parasite’s evasion of detection and elimination.

An archetype anti-rosetting vaccine against severe disease is being developed as is a receptor-based drug, the latter together with a recently funded KI AB company, Dilafor AB. Dilaforette

Our work is carried out in close co-operation with professors Fred Kironde and Florence Mirembe at Makerere University and Tomas Egwang, Medical Biotech Labs, all in Kampala, Uganda. Scientists from China, Denmark, France, Germany, Japan, Papua New Guinea, Spain, Sweden and USA are also part of our network of collaborators.

Projects

Burkitt's lymphoma: PfEMP1 and Epstein-Barr virus

Burkitt's lymphoma, an aggressive B cell malignancy, is the most common cancer in children in the tropics accounting for 30-70% of childhood cancers in Africa.

In the present project we analyze the mechanisms by which chronic malaria infection affects the regulation of the B cell compartment and the relationship between EBV and B-lymphocytes. We have found that that malaria may join forces with EBV to heighten the risk for Burkitt's lymphoma. P. falciparum is highly mitogenic for B cells. The P. falciparum erythrocyte membrane protein 1 (PfEMP1) contains a cysteine-rich inter-domain region 1± (CIDR1±) that acts as a potent activator and stimulator of B lymphocyte proliferation (Chene et al PLoS Path, 2007). It is particularly active against memory B cells (Donati et al J.Immunol, 2005), which, coincidentally, are where EBV persists in vivo.

We hypothesize that in individuals chronically stimulated by the malaria parasites the B-cell compartment is protected from early death, apoptosis. In addition, malaria parasite stimulation disturbs the control of EBV latency leading to an increase in virus production. The combination of the two effects increases the pool of B cells with the potential to oncogenic changes that could lead to the development of BL.

Genome deletions and duplications in P.falciparum

In this project we study the presence of gene copy number polymorphisms (CNPs). Further analysis of the impact of discovered genetic differences and the underlying mechanisms is likely to generate a better understanding of the biology and the virulence of the malaria parasite.

The extent to which duplications and deletions occur in the Plasmodium falciparum genome, outside of the subtelomeres, and their contribution to the virulence of the malaria parasite is not known. We have recently found CNPs covering 82 genes, the most extensive spanning a cumulative size of 110 kilobases (Ribacke et al, MBP 2007). CNPs were identified in both laboratory strains and fresh clinical isolates using a 70-mer oligonucleotide microarray in conjunction with fluorescent in situ hybridizations and real-time quantitative PCR. The CNPs were found on all chromosomes except on chromosomes 6 and 8 and involved a total of 50 genes with increased copy numbers and 32 genes with decreased copy numbers relative to the 3D7 parasite. The genes, amplified in up to six copies, encode molecules involved in cell cycle regulation, cell division, drug resistance, erythrocyte invasion, sexual differentiation and unknown functions. These together with previous findings, suggest that the malaria parasite employs gene duplications and deletions as general strategies to enhance its survival and spread.

The complete genome sequence of the human malaria parasite Plasmodium falciparum has revealed a block of genes (³ 10 kilobases in size) which has been duplicated and transposed on multiple subtelomeres (Mok et al, submitted). We found both inter- and intragenic regions of these SD are highly conserved across species, except for a hypervariable region encoding a surface-exposed loop of the PFMC-2TM. The existing numbers of this SD per genome is here shown to be variable in between different parasite lines, suggesting variable expansion and/or deletions of existing duplicons. The ubiquity and uniqueness of the SDs in the P. falciparum subtelomeres, and the nature of genes within suggests an important role in plasmodia speciation.

Heparan sulphate and other sequestration receptors

Project member: Anna Vogt

PfEMP1-species of infected erythrocytes of children with severe malaria frequently bind to the host receptor heparan sulfate present on both endothelial cells and erythrocytes (Barragan et al Blood 2000, Heddini et al, Inf. Immun. 2001, Vogt et al, Blood, 2003). 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.

We recently discovered that de-polymerized heparin that lacks anticoagulant activity blocks up to 80% of infected erythrocytes from binding in the micro-vasculature and releases already sequestered parasites into circulation (Vogt et al PloS Path, 2006). We suggest the modified heparin to be a promising candidate for adjunct therapy in severe malaria and are presently developing the substance it into a drug together with a recently funded KIAB company

Dilaforette

Immunoglobulin-binding and placental malaria

The harmful effects of pregnancy-associated malaria are engendered by the heavy sequestration of Plasmodium falciparum parasitized RBCs in the placenta. It is well documented that this process is mediated by interactions of parasite-encoded variant surface antigens and placental receptors. A P. falciparum erythrocyte membrane protein 1 variant, VAR2CSA, and the placental receptor chondroitin sulfate A (CSA) are currently the focus of PAM research.

We have previously shown a role for immunoglobulins (IgG and IgM) from normal human serum as additional receptors in placental sequestration and in resetting (Scholander et al, Nature Med 1996; Flick et al, Science 2001). In this project we are studying the phenoytypes of parasites eluted from the placenta and parasites in vitro and found (i) that CSA and non immune IgG-IgM binding are linked phenotypes of in vitro-adapted parasites, (ii) that a VAR2CSA variant shown to bind CSA also harbors IgG- and IgM-binding domains (DBL2-X, DBL5-!, and DBL6-!), and (iii) that IgG and IgM binding and adhesion to multiple receptors (IgG/IgM/HA/CSA) rather than the exclusive binding to CSA is a characteristic of fresh Ugandan placental isolates. These findings are of importance for the understanding of the pathogenesis of placental malaria and have implications for the ongoing efforts to develop a global PAM vaccine.

Novel Ag332 at the infected erythrocyte surface?

In this project we are studying a novel part of the Ag332 which we recently identified (Moll et al PLoS One, 2007) using biochemical and molecular tools. An open reading frame representing a hitherto unknown second exon of the Pf332 gene that encodes a cysteine-rich polypeptide with a high degree of similarity to the Duffy-binding-like (DBL) domain of the erythrocyte-binding-ligand (EBL) family was found (Moll et al, PLoS One 2007).

The sequence of this DBL-domain is conserved and expressed in all parasite clones/strains investigated. In addition, the expression level of Pf332 correlates with proliferation efficiency of the parasites in vitro. Antibodies raised against the DBL-domain are able to reduce the invasion efficiency of different parasite clones/strains. Analysis of the DBL-domain revealed its ability to bind to uninfected human RBC, and moreover demonstrated association with the iRBC surface. Thus, Pf332 is a molecule with a potential role to support merozoite invasion. Due to the high level of conservation in sequence, the novel DBL-domain of Pf332 is of possible importance for development of novel anti-malaria drugs and vaccines.

PfEMP1-motifs, rosetting and the pathogenesis of severe malaria

We have previously identified virulence-associated markers of the parasite such as resetting and giant resetting (Udomsangpetch et al JEM, 1989; Carlson et al Lancet, 1990; Carlson et al PNAS 1990). In this project we have been studying var-RNA of Ugandan children with mild or severe malaria in cooperation with professors Fred Kironde and Tom Egwang and other scientists at Makerere University-MBL. We have been using a novel method for sub-sectioning region alignments into homology areas (MOTIFF) developed in the laboratory of Björn Andersson at Karolinska Institutet and we found specific PfEMP1 amino acid motifs to be associated with severe disease. The method is applicable to any family of variant proteins and the results suggest that certain PfEMP1 species are predisposed to inducing severe malaria (Normark et al, PNAS, 2007).

RIFINS/SURFINS at the surface of the infected erythrocyte and merozoite

The rif genes, which encode RIFIN proteins, is the largest family in the P. falciparum genome with ~150 copies per haploid genome. They are small two-exon genes with a conserved domain architecture. Our group, as well as others, has shown that RIFIN proteins are exported from the parasite to the infected cell surface (Helmby et al, Inf.Immun 1992, Fernandez et al, JEM, 1999). We have recently determined that the RIFIN protein family can be subdivided into two major groups (Joannin et al. submitted to BMC Genomics). In addition to demonstrating differential developmental regulation and localization of these sub-families (Petter et al, in press Molecular and Biochemical Parasitology 2007), we have also predicted that these proteins have undergone a functional shift (Joannin et al. submitted to BMC Genomics).

SURFINs are encoded by a family of 10 surf genes, including a predicted pseudogene, located within or close to the sub-telomers of five of the chromosomes. SURFINs show structural and sequence similarities with exported, surface exposed proteins (PvSTP1, PkSICAvar, PvVIR, Pf332, PfEMP1) of several plasmodium species. SURFIN4.2 has been found co-transported with PfEMP1 and RIFIN to the IE surface but also accumulated in the parasitophorous vacuole. In released merozoites SURFIN4.2 was present in an amorphous cap at the parasite apex where it may be involved in the invasion of erythrocytes (Winter et al JEM 2005).

By exposing shared polymorphic antigens on IEs and merozoites the parasite may coordinate the antigenic composition of these attachment-surfaces during growth in the bloodstream.

Vaccine against severe malaria

Building on the sequencing of var genes of children with severe or mild malaria (project: PfEMP1-motifs, rosetting and the pathogenesis of severe malaria) we have in parallel studies created a prototypic anti-severe malaria vaccine composed of a PfEMP1-molecule that harbors such severe malaria amino-acid motifs (Chen et al, Vaccine 2004; Pettersson et al, I&I 2005). Adhesion of infected erythrocyte in the micro-vasculature and the subsequent development of severe malaria is prevented by immunizing animals, including primates, with the PfEMP1- vaccine prior to challenge (Moll et al I&I 2007).

Antibodies are an important component of acquired protective immunity

In our group, we are trying to understand which functions of antibodies that are important for protection against malaria. Basic understanding of how these antibodies work is imperative for creating a functioning vaccine against malaria. We are studying the affinity of antibodies directed against different merozoite antigens, as well as the invasion inhibitory effect, and correlating this to protection. We are also investigating different invasion pathways in wild isolates of parasites, and correlating this to symptoms and severity of disease. We have before developed a method for measuring invasion inhibitory antibodies, and we have also shown that variation in use of invasion pathways (involving the merozoite antigens EBA and Rh proteins) mediates evasion of inhibitory antibodies.

Variation in use of erythrocyte invasion pathways by Plasmodium falciparum mediates evasion of human inhibitory antibodies.
Persson KE, McCallum FJ, Reiling L, Lister NA, Stubbs J, Cowman AF, et al
J Clin Invest 2008 Jan;118(1):342-51

Development and optimization of high-throughput methods to measure Plasmodium falciparum-specific growth inhibitory antibodies.
Persson KE, Lee CT, Marsh K, Beeson JG
J Clin Microbiol 2006 May;44(5):1665-73

Investigation of functionally important antibodies against malaria

Project members: Kristina Persson, Sreenivasulu Reddy, Hodan Ahmed Ismail, Caroline Rönnberg, Allan Lugaajju, Tijani Muyideen Kolapo.

The merozoite form of P. falciparum invades red blood cells and multiplies within the cell, finally causing rupture of the red blood cell with release of daughter merozoites back into the circulation after 48 hours. Those who live in malaria-endemic areas and do not die from the disease at a young age, eventually develop immunity, but only slowly and after repeated exposure. Antibodies are an important component of acquired protective immunity.

In our group, we are trying to understand which functions of antibodies that are important for protection against malaria. Basic understanding of how these antibodies work is imperative for creating a functioning vaccine against malaria. We are studying the affinity of antibodies directed against different merozoite antigens, as well as the invasion inhibitory effect, and correlating this to protection. We are also investigating different invasion pathways in wild isolates of parasites, and correlating this to symptoms and severity of disease. We have before developed a method for measuring invasion inhibitory antibodies, and we have also shown that variation in use of invasion pathways (involving the merozoite antigens EBA and Rh proteins) mediates evasion of inhibitory antibodies. We are also investigating the interaction between B-cells and malaria parasites.

Plasmodium falciparum line-dependent association of in vitro growth-inhibitory activity and risk of malaria.
Rono J, Färnert A, Olsson D, Osier F, Rooth I, Persson KE
Infect Immun 2012 May;80(5):1900-8

High affinity antibodies to Plasmodium falciparum merozoite antigens are associated with protection from malaria.
Reddy SB, Anders RF, Beeson JG, Färnert A, Kironde F, Berenzon SK, et al
PLoS One 2012 ;7(2):e32242

Erythrocyte-binding antigens of Plasmodium falciparum are targets of human inhibitory antibodies and function to evade naturally acquired immunity.
Persson KE, Fowkes FJ, McCallum FJ, Gicheru N, Reiling L, Richards JS, et al
J Immunol 2013 Jul;191(2):785-94

Variation in use of erythrocyte invasion pathways by Plasmodium falciparum mediates evasion of human inhibitory antibodies.
Persson KE, McCallum FJ, Reiling L, Lister NA, Stubbs J, Cowman AF, et al
J Clin Invest 2008 Jan;118(1):342-51

Host-parasite interactions in schistosomiasis and virtual microscopy

The excretory-secretory system of intravascular blood flukes, schistosomes is studied as a target for the host immune response. Glycans at the parasite surface have been observed to originate from the parasite protonephridial system, a sophisticated phylogenetically ancient organ. The protonephridial system also seems to be involved in the metabolization of host serum immunoglobulins as part of an unknown transportation system.

Future studies aim at further characterization of the schistosome protonephridia using novel immunohistochemical light and ultrastructural markers.

Studies aiming at education and an improved parasitological diagnostic service at the basic level of health care in developing countries have been initiated (see link below). The idea is to generate a digitalized library of parasitological specimens freely available at the www and consisting of entire specimens scanned in high magnification and permitting viewing in the x, y and z plane at different magnification.

Project Leaders

Anders Björkman Project

Qijun Chen Professorship in Beijing

Akira Kaneko Project

Manuel Patarroyo Project

Ulf Ribacke Project

Group Members