Structural analysis of the CSA binding interactions involved during pregnancy associated malaria

Final Report Summary - PREMALSTRUCT (Structural analysis of the CSA binding interactions involved during pregnancy associated malaria)

Executive summary:

Each year, 200 to 400 million clinical cases of malaria are reported globally, causing around 1 million deaths, largely in the sub-Saharan continent. Plasmodium falciparum and, to a much lesser extent, P. vivax are the main causes of disease and death from malaria. An important difference between P. falciparum and other human malaria parasites is the way in which P. falciparum modifies the surface of the erythrocyte so that asexual parasites can adhere to host cells.

Women become more susceptible to severe Plasmodium falciparum infections during their first pregnancy. Pregnancy-associated malaria (PAM) is coupled with massive accumulation of parasitised erythrocytes in the placental intervillous blood spaces, contributing to disease and death in pregnant women and developing infants. Prevention may be possible by vaccinating women before their first pregnancy. Adhesion of P. falciparum-infected erythrocytes (PE) to placental chondroitin-4-sulfate (CSA) has been linked to the severe disease outcome of PAM.

After one or two pregnancies, transcendent antibodies that recognise placental PEs from different geographic regions develop and correlate with protection from malaria. Antibodies against CSA-binding parasites are associated with maternal malaria resistance after multiple pregnancies and also block CSA-binding of placental isolates from different parts of the world, demonstrating the concept of a transcending immune response to the P. falciparum ligands that mediate adhesion to CSA.

These findings suggest that the surface molecule(s) expressed by placental variants have conserved antigenic determinants, thus spurring efforts to characterise them in order to induce protective antibodies by vaccination.

Recent evidence strongly suggests that var2CSA, a member of the P. falciparum Erythrocyte Membrane protein 1 (PfEMP1) family, may play an important role in PAM and immunity. Although var2CSA is the main candidate for a pregnancy malaria vaccine, experimental evidence implies that antigenic polymorphism and the lack of a small animal in vivo experimental model may pose a challenge for vaccine development.

It is vital that research deciphers the molecular basis for the CSA binding to the parasite ligands in order to define the common features within the different CSA-binding domains and the cross-reactive epitopes that are likely to be the targets of natural protective antibodies. This knowledge will be helpful in the design of novel PfEMP1-CSA-based antigens capable of inducing broad and potent neutralising antibodies to a wide variety of strains, and in identifying molecules with inhibitory capacity that could be considered for therapeutic strategies as anti-adhesive drugs.

Over three years, a major aim of this project was to provide knowledge to scientific groups working in the PAM vaccine and therapeutic fields.

Among the main achievements figure:

- the recombinant expression of a functional full length extracellular region of var2CSA (300 kDa protein) that binds to CSPG / CSA with very high affinity and specificity;
- the evidence that several VAR2CSA domains are required to form the complete CSA-binding site rather than several independent sites being contributed by single domains;
- the high resolution structure of the DBL6-FCR3 domain and the DBL3X-DBL4-3D7 double domains;
- the low resolution molecular envelope of the full-length DBL1X-DBL6 recombinant protein obtained by analytical ultracentrifugation and Small angle X-ray scattering (SAXS);
- the identification of the minimal CSA-binding site of VAR2CSA from the FCR3 and 3D7 alleles;
- the stoichiometric requirements for binding of P. falciparum-infected erythrocytes to CSA;
- the creation of a small animal model for in vivo investigations of host-parasite interactions in pregnancy associated malaria.

These achievements have direct implications for the development of a PAM vaccine and drugs.

Project Context and Objectives:

The goal of the PREMALSTRUCT project is to provide a molecular basis for understanding the specificity of placental P. falciparum isolates for CSA binding, which leads to PAM via sequestration of infected red blood cells (iRBC) in the placenta. We aim to elucidate the structural correlates of CSA binding by PfEMP1 as well as of protective B-cell epitopes in this parasite adhesin. A large body of evidence indicates that immunity to PAM arises from antibodies that inhibit the binding of placental isolates to placental proteoglycans carrying CSA. The VAR2CSA variant of the PfEMP1 appears to be the exclusive member of this parasite adhesin family. Although VAR2CSA is polymorphic, cross-reactive B-cell epitopes appear to be responsible for natural immunity acquired after several pregnancies by women living in malaria-endemic regions. Accordingly, we aim to define the common features within the different CSA binding domains of VAR2CSA and to characterise the cross-reactive epitopes that are the likely targets of natural protective antibodies. From these results, we expect to establish a firm molecular basis for the development and optimisation of vaccine and therapeutic strategies by providing upstream knowledge from three-dimensional structure of VAR2CSA and relating this information to CSA specificity and to the distribution of protective epitopes. This knowledge will not only be useful in the design of CSA-binding PfEMP1 immunogens capable of inducing broad and potent neutralising antibodies to a wide variety of strains, but also to identify molecules with inhibitory capacity that could be considered for therapeutic strategies as anti-adhesive drugs.

The PREMALSTRUCT consortium expect to:

1. Solve the three-dimensional structure of different var2CSA CSA-binding DBL domains in order to:
- understand their binding and their specificity for CSA;
- define the common features within the different CSA binding domains;
- analyse the spatial distribution of dominant B-cell epitopes and cross-reactive epitopes;
- identify small molecules that prevent and/or reverse binding of P. falciparum-infected erythrocytes to CSA.

2. Define the stoichiometric and spatial requirements for a productive CSA / PfEMP1 interaction under both static and flow conditions.
3. Create an in vivo model that would permit small animal validations of vaccination and small molecule inhibitors strategies.

To implement this research, we have built a consortium around the core group of six internationally renowned institutions (four EU countries and one ICPC country) and one SME. All the beneficiaries have a long tradition in malaria research and are distinguished for their technological advances in the field of molecular and structural biology.

Project results:

As originally proposed, the different consortium members are involved in 5 scientific different Work package (WP)s and one management WP. The highly efficient work performed by the different PREMALSTRUCT partners has led to considerable progress towards our planned objectives and deliverables, which are described in annex I. The PREMALSTRUCT consortium has made significant contribution through key discoveries on the molecular nature of the interaction between Plasmodium falciparum infected erythrocytes and CSA.

WP1 - Expression of functional CSA-binding DBL domains
WP leader: G. BENTLEY (Beneficiary 1 - Institut Pasteur (IP))

Summary

The goal of WP1 was to generate recombinant proteins in mg amounts as well as monoclonal antibodies against CSA binding domains for use in structural, functional and immunological studies. During the project, a large number of proteins have been expressed in mg amounts and mAbs were successfully obtained against all the DBL domains composing the 3D7 var2CSA variant. Due to novel results obtained within the PREMALSTRUCT consortium, it has been decided to include multiple DBL domains and full length var2CSA constructs in the list of proteins to be express for structural determination using other methods than crystal diffraction, such as SAXS and EM. These slight adjustments were proposed by the WP leader in agreement with the project coordinator and the SAC members (Prof. Alister CRAIG of the Liverpool School of Tropical Medicine and Dr Qijun CHEN of the Karolinska Institutet). Although MAbs directed against the DBL1X, DBL2X, DBL4, DBL5 and DBL6, are only recognising the homologous variant, MAbs targeting the DBL3X domain cross-react with heterologous DBL3X domains. Furthermore, the Mabs targeting the DBL3X domains inhibit the interaction between the full length var2CSA recombinant protein and decorin.

1. To provide recombinant correctly folded CSA-binding DBL domains in mg quantities for structural, functional and immunological assays

The production of correctly folded DBL domains in milligram amounts is an essential step for functional characterisation and crystallographic studies. Previous studies indicate that four of the six domains of var2CSA (DBL2X, DBL3X, DBL5, DBL6) and the DBL3 domain of var1CSA have affinity for CSA. One major concern was to identify the correct sequence delimiting the structural domain of the DBL motif. An incorrect choice of sequence limits can lead to incorrectly folded protein or give a product with flexible N and / or C-termini that can compromise success in crystallisation trials. The systematic search for a stable, well-defined structural domain was achieved by adapting incremental gene truncation procedures to systematically alter the extremities of the DBL constructs. Accordingly, it was necessary to test several constructs to obtain soluble recombinant proteins.

An important portion of the work was dedicated to the production of DBL domains using different expression systems such as E. coli, P. pastoris, HEK293 and baculovirus / insect cells (WP1). A large number of proteins have been expressed in mg amounts.

Recombinant domains were characterised by biophysical and biochemical methods to ensure that these proteins were produced in a homogeneous native state that preserved their biological function:

- All domains produced in HEK cells, E. coli and P. pastoris (see deliverable D1.2) were subjected to limited proteolysis; these analyses gave a small number of bands, consistent with a homogeneous, correctly folded protein.
- Circular dichroism (CD) spectra were measured on all E. coli and P. pastoris protein products; CD in the far UV confirmed the expected high-helical content while CD in the near UV showed that they had a stable, homogeneous tertiary structure.
- Solid phase binding studies were performed to analyse the specificity of the recombinant domains for CSA by comparing fixation to various sulphated oligosaccharides.
- Binding constants of the recombinant domains for CSA and other sulphated oligosaccharides were determined by surface plasmon resonance techniques.

2. To generate MAbs directed against CSA-binding DBL domains for crystallographic studies made as complexes with Fab fragments

Recombinant full length VAR2CSA protein produced in HEK293 cells was used to produce monoclonal antibodies against the different DBL domains. Full length 3D7 VAR2CSA protein was used for immunisation of mice to generate MAbs using standard methods.

MAbs were screened by ELISA against the full length var2CSA as well as homologous and heterologous var2CSA derived recombinant DBL domains produced in WP1. MAbs were then screened for their capacity to inhibit the interaction between the full length var2CSA recombinant protein and decorin immobilised on 96 wells ELISA plates.

Following the screening process, MAbs were successfully obtained against all the DBL domains composing the 3D7 var2CSA variant and are of the IgG1 isotype. Although MAbs directed against the DBL1X, DBL2X, DBL4, DBL5 and DBL6, are only recognising the homologous variant, MAbs targeting the DBL3X domain cross-react with heterologous DBL3X domains. Furthermore, the Mabs targeting the DBL3X domains inhibit the interaction between the full length var2CSA recombinant protein and decorin.

WP2 - Structural analysis of CSA-binding DBL domains
WP leader: A. SHARMA (Beneficiary 2 - International Centre for Genetic Engineering and Biotechnology (ICGEB))

Summary

The goal of WP2 is to provide structural data of VAR2CSA domains that lead to functional insights into the specific, high affinity binding to CSA by this PfEMP1 variant. This is achieved from three-dimensional structures determined by X-ray crystallographic techniques. It thus depends on the successful production of homogeneous recombinant domains (goal of WP1) in quantities adequate for extensive crystallisation trials that would enable diffraction measurements and subsequent structure determination.

During the project, we have successfully crystallised and determined the structure of the DBL6-FCR3 domain and the DBL3X-DBL4-3D7 double domain.

During this project, we found strong evidence that several VAR2CSA domains are required to form the complete CSA-binding site rather than several independent sites being contributed by single domains (i.e. an avidity effect). As crystallisation of multi-domains presents a formidable task, we studied these recombinant proteins by SAXS, as well. Using this low-resolution technique, we analysed the molecular envelope of the full-length DBL1X-DBL6 and DBL4-DBL6 proteins.

FCR3-DBL6

Crystals were grown by vapour diffusion with initial buffer conditions found by robotic screening. Final conditions were optimised by finer screening around this initial hit.

The structure of FCR3-DBL6 was determined by molecular replacement using the structure of the 3D7-DBL6 (PDB ID 2wau, published outside this consortium) as a search model. The structure of FCR3-DBL6 shows significant differences to the structure of the homologous 3D7-DBL6 domain (Khunrae et al., J Mol Biol, 393:202, 2009, determined to 3.0 Å), mainly at the N-terminal region. This is because one of the cysteines in the 3D7 domain was erroneously assigned as being in the reduced state (i.e. no cystine bridge) and accordingly was mutated to serine to avoid potential covalent aggregation. By contrast, we have adopted the strategy of blocking unpaired cysteines by cystamine to maintain the wild-type sequence but at the same time to avoid coupling of domains by intermolecular disulphide bridges. We have thus avoided an erroneous assignment of cysteines and all native disulfide bridges were able to form. The N-terminus is thus stabilised by a disulfide bridge and its structure is well defined. The significantly higher resolution of the FCR3 structure has led to a more precise definition of the protein atomic coordinates.

FCR3-DBL3X-DBL4

Crystals of FCR3-DBL3X-DBL4 were obtained by vapour diffusion by manual screening with a limited number of buffer conditions. Two types of crystals were obtained under the same crystallisation conditions:

The structure was determined for the type 2 crystal form by molecular replacement using the high-resolution structure of FCR3-DBL3X (PDB entry 3bqk). Crystals of this form were of limited quality but diffraction data could be measured to 2.8 Å resolution.

Molecular replacement allowed the DBL3X domain to be positioned in the unit cell, but no solution was apparent for the DBL4 moiety, indicating significant structural differences of the latter domain with respect to the former. Electron density for the DBL4 domain could be identified in the Fourier maps and the structure determination is currently progressing by alternate cycles of model building and refinement.

Additional single and double domains were expressed in E.coli. All were purified as soluble proteins and used in protein crystallisation trials under different conditions using robotic or manual crystallisation assays as appropriate.

Structural studies by SAXS

The main objective of this WP is to define the structure of the CSA-binding sites on VAR2CSA domains. During this project, however, results from the Consortium showed that the binding site is formed by several domains in concert (see WP3).

As crystallisation of multi-domain VAR2CSA constructs poses several technical difficulties, we also approached question using SAXS techniques. Thus, we have determined the shape of the entire extracellular domain (DBL1X-DBL6) and the DBL4-DBL6 triple domain by SAXS, showing that the protein is compact, with an overall length of 18.5 nm (if it were extended, it would be about 43.8 nm in length). The SAXS data are coherent with AUC data for both DBL4-6 and DBL1X-6.

WP3 - Characterisation of the CSA-binding sites
WP leader: A. SALANTI (Beneficiary 3 - University of Copenhagen (UCPH))

Summary

WP3 utilises proteins produced in WP1 with the aim of identifying regions of VAR2CSA displaying affinity for CSA. WP3 complements WP2, which aims to identify interactions using structural techniques (crystallography). This WP should define those parts of VAR2CSA comprising the minimal binding region followed by a more detailed analysis of key residues participating in the interaction with CSA. During this project, we found strong evidence that several VAR2CSA domains are required to form the complete CSA-binding site rather than several independent sites being contributed by single domains. To define more precisely the minimal regions of the full-length VAR2CSA that are necessary for specific and high affinity interaction, we expressed various truncated versions of var2CSA in insect cells and HEK293 cells, and used biosensor technology and SPR to examine their binding properties (WP3). Our binding studies provide compelling evidence that the minimal CSA-binding site of VAR2CSA from the FCR3 and 3D7 alleles lies within the N-terminal region, i.e. DBL1X-DBL3X, with the DBL2X-CIDRpam being the core component. These results have important consequences for the development of an effective vaccine and therapeutic inhibitors.

Task 1: Identification of minimal binding domains within CSA-binding DBL domains using biochemical methods

All recombinant proteins produced in WP1 were screened for CSA binding in an ELISA-based system. CSA binding capacity was confirmed for DBL2, DBL3 and DBL6; however, we demonstrated that a number of DBL domains of non-VAR2CSA origin showed that CSA-binding is not exclusive to VAR2CSA DBL domains. Furthermore, we found that the VAR2CSA DBL domains, as well as other DBL domains produced as controls, also bind heparan sulphate and other sulphated carbohydrates. Our data suggest that the ability of single domains to bind CSA does not reflect the functional behaviour of the complete VAR2CSA adhesin, placing previously published conclusions in doubt. It was therefore not appropriate to map minimal CSA-binding sites within the single domains as the specificity and affinity of the binding of these sites was low.

We therefore produced the full-length VAR2CSA protein in insect cells and HEK293 cells, and showed that this protein bound to CSPG/CSA with very high affinity and specificity, (Khunrae et al, JMB; Srivastava et al, PNAS). To define more precisely the minimal regions of the full-length VAR2CSA that are necessary for specific and high affinity interaction, we expressed various truncated versions of var2CSA in insect cells and HEK293 cells, and used biosensor technology and SPR to examine their binding properties.

Our binding studies provide compelling evidence that the minimal CSA-binding site of VAR2CSA from the FCR3 and 3D7 alleles lies within the N-terminal region, i.e. DBL1X-ID1-DBL2X-CIDRPAM-DBL3X, with the DBL2X-CIDRpam being the core component. The DBL2X-CIDRpam protein does not fully reproduce the specific, high affinity CSA binding of the full-length protein, but when this core complemented with either the DBL1X domain at the N-terminal end, or the DBL3X domain in the C-terminal end, or with both these domains, the properties of the full-length protein are largely restored. It is possible that these flanking domains stabilise the binding core without directly participating in the CSA binding. Alternatively, DBL1X and DBL3X exhibit residues that are directly involved in the CSA binding, which is enhanced by the presence of these domains.

Taken together, our results suggest that the high-affinity, CSA-binding site lies within DBL1X-3X moiety and that interaction between these domains and the domains of the C-terminal moiety (DBL4-DBL6) are important for its stability.

Task 2 Expression of chimeric DBL domains and deletion constructs to map the CSA binding site and task 3 Mapping binding residues within CSA-binding DBL domains

The initial objectives of these tasks have become inappropriate because during the course of this project we have shown that the affinity and specificity of var2CSA for CSA depend on the presence of several domains. In particular, the N-terminal region of var2CSA (DBL1X-DBL3) comes closest in reproducing the properties of the complete extracellular region.

Conclusion

Several key articles have been produced based on the work done in WP3. In particular we have shown that single VAR2CSA do not as expected bind with high specificity or affinity to CSA (Rezende et. al., 2009 and Dahlbäck et. al., 2010). The participants of WP3 accordingly re-organised the research strategy with the aim of defining novel parts of VAR2CSA that binds to CSA with the desired specificity and affinity. In 2010 we published two articles describing the production and characterisation of full length VAR2CSA where it was shown that full length protein does bind to CSA with high affinity and specificity (Khunrae et. al., 2010, Srivastava et. al., 2010). Instead of starting to map residues as described in task i and ii we produced panels of N and C-terminal truncated versions of the full length protein and by this method we successfully mapped the CSA binding site within the N-terminal region of var2CSA (DBL1X-DBL3X), these data were presented in two articles (Dahlbäck et. al., 2011 , Srivastava et. al., 2011). In conclusion the work done in WP3 has been very successful and generated novel and interesting data that was published in highly esteemed international journals.

WP4 - Characterisation of the PfEMP1-CSA interaction and identification of molecules preventing adhesion
WP leader: M. LANSER (Beneficiary 4 - University of Heidelberg (UKL-HD))

Summary

Good progresses were made in WP4 on the stoichiometric requirements for binding of P. falciparum-infected erythrocytes to CSA and on the identification of anti-adhesive compounds. Indeed, biophysical experiments using reconstituted membranes with defined receptor density have provided detailed information of the physical parameters such as the number of interactions as well as the binding strength between malaria iRBC to CSA. Virtual screenings were performed to identify compounds that mimic CSA. Several modelling experiments were performed using in most of the cases the structural information of a tetramer carbohydrate derivative which mimics the structure of the portion of the CSA molecule responsible for the binding of the infected erythrocytes (expressing Var2CSA on the surface). These experiments provided several hints on the structure of molecules which could be purchased or synthesised during the project and were used in vitro to search an inhibitor of the interaction between the IE with CSA. As a result, one hit emerged from these screening campaigns, SC85516, which inhibited the interaction at relatively high concentrations (between 100 micromole to 1 mM).

Characterisation of the PfEMP1-CSA interaction

The strategy was to establish a quantitative model of placenta tissue surface displaying CSA A at a defined surface density. The core part of this system is a planar lipid bilayer membrane deposited on planar substrates. By doping biotinylated lipid anchors, these can be used to couple biotin-tagged CSA via neutravidin cross-linkers. This setup allows us to control the average intermolecular distance between CSA molecules between 3.4 and 54 nm in nm accuracy.

The proof of principle was demonstrated by studying the adhesion of red blood cells (RBCs) at an average CSA distance of 5.4 nm. After one hour incubation on static conditions, we confirmed the specific adhesion of infected RBC. In fact, the adhered cells cannot be removed by rinsing the setup. Healthy RBC and RBC infected with the FCR3 VARCSA show no detectable adhesion on the surface.

In order to determine the influence of on the cell adhesion, the lateral density of adherent red blood cells were counted at various . In the case of cells that are 28 h after the infection (trophosoites), a sharp increase in the density of the adherent cells can be observed between equal to 10.8 nm and equal to 5.4 nm by a factor of 5. RBC after 34 h of infection (schisonts) undergoes even a sharper transition by changing the intermolecular distance from equal to 34.2 nm and equal to 10.8 nm. The obtained results indicate that the critical lateral density CSA required for causing firm adhesion is dependent on the development of parasite infections.

To further investigate the adhesion-induced deformation of red blood cells, the three-dimensional height profiles of infected / uninfected cells near the surface were reconstructed from the confocal microscope image stacks. The bottom surface of an infected cell adhered on the membrane with a high CSA density can no longer retain the concave shape of a natural cell and is flattened due to the strong adhesion to the membrane surface. The degree of cell deformation shows a monotonic increase according to the increase of the lateral density of CSA molecules.

For the visualisation of shape and contact area on the bottom side of the inf RBC was the Reflection interference contrast microscopy (RICM) technique used to reconstruct the absolute height distance between an object and the substrate.

To determine quantitatively the binding strength between the model placental surfaces displaying CSA and the iRBC, a new assay utilising shock waves induced by picosecond laser pulses was developed by Dr H. Yoshikawa in the Tanaka lab (Yoshikawa, et al., J. Am. Chem. Soc. 2011). The critical pressure for cell detachment can be used as a quantitative measure to evaluate the cell adhesion strength without tracer particles.

A monotonic increase in the peak position P* from 0.75 to 2.3 MPa was observed according to the decrease in the average distances between CSA from 54 to 5.4 nm, indicating that infected erythrocytes sensitively detect the surface density of CSA within nm accuracy. On the other hand, a change in from 5.4 to 3.4 nm causes no increase in P* suggesting the saturation due to the condensation of polymers into the 'brush' regime.

In the later infection stage (schizont), the infected cells show a significant increase in P*(data not shown). The maximum P* (~ 3.5 MPa) could be reached at equal to 5.4 nm (P*), which is 1.5 times larger than the corresponding value for trophosoites (P* approximately 2.3 MPa). This finding can be attributed to an increase in the number of knobs on the host cell surface during the parasite development.

The combination of the quantitatively functionalised cell surface models and the new physical tool demonstrated that small changes in the average CSA distance caused a significant increase in the cell adhesion strength. Such a technical platform can be used to determine the time evolution of cell adhesion strength according to the development of parasites.

Identification of molecules preventing adhesion

During the first part of the project, two virtual screenings were performed, one by alignment using a short analogue of CSA (a tetramer) and one after docking into the putative binding pocket of domain 3 of Var2CSA (DBL3x). After the two screenings, several compounds were purchased and tested in vitro as inhibitor of the interaction between infected erythrocytes and CSA. As a result, one hit emerged from these screening campaigns, SC85516, which inhibited the interaction at relatively high concentrations (between 100 micromole to 1mM).

Further work performed was based on the structure of this compound, since the structural details of the interaction between Var2CSA and CSA were not available.

A virtual library of millions of pyrimidin triones was built up by computational method. This virtual library was aligned with the standard oligosaccharide tetramer analog of CSA, using the proprietary software Propose&174; and 4SCAN&174;. The molecules with the highest similarity score after the experiment were selected and synthesised. The syntheses attempted were not successful in producing the final product in sufficient amount and purity and were abandoned.

In the search of new classes of compounds, new modelling experiments were performed using the CSA analogue tetramer as 'ligand' template: Search 4SC's database of commercial compounds (ca. 5 million) for sulfate containing compounds and align them with the template. Using the new 4SC's software (PepDesign&174;), build-up a virtual library of tripeptides containing sulfo-serine and 'non-natural' amino acids and align them with the template.

The rational of these new experiments was the recognition that sulphate groups play an important role in the binding between Var2CSA and CSA (S.V. Madhunapantula, et al., The Journal Of Biological Chemistry, 282, pp. 916-92, 2007).

In conclusion, several modelling experiments were performed using in most of the cases the structural information of a tetramer carbohydrate derivative which mimics the structure of the portion of the CSA molecule responsible for the binding of the infected erythrocytes (expressing Var2CSA on the surface). These experiments provided several hints on the structure of molecules which could be purchased or synthesised during the project and were used in vitro to search an inhibitor of the interaction between the IE with CSA. In the future, when more detailed structural information about this interaction will be available, docking experiments performed on the binding pocket of the protein should produce molecules capable of inhibition of this interaction.

WP5 - Creation of a small animal model for in vivo investigations
WP leader: B. FRANKE (Beneficiary 5 - University of Leiden (LUMC))

Summary

The goal of WP5 is to create a small animal model for investigations of host-parasite interactions in pregnancy associated malaria (PAM). The aim is to use the rodent parasite Plasmodium berghei to analyse in vivo interactions between P. falciparum var2CSA domains, expressed on the surface of iRBC and CSA. By proteomic, bioinformatic and molecular approaches we have identified a set of blood stage P. berghei proteins that are exported into the host cell cytoplasm and two proteins, Pbfam1.1 and NSC1 that are present in the iRBC membrane. These parasite membrane proteins are candidates to use as a tool for the expression of heterologous domains on the iRBC surface. We showed that fusions of Pbfam1.1 with a P. falciparum CD36 binding CIDR1 domain and with DBL1-6 domains of var2CSA create P. berghei parasites that are able to specifically bind to receptors in in vivo or in vitro assays. Variants of this system can be used and implemented to test drugs and vaccine candidates developed within the consortium and by many researchers working in diverse fields in malaria research.

Identification and analysis of proteins present on the surface of P. berghei iRBC

In order to identify P. berghei iRBC surface proteins, we have executed several complementary proteomic approaches. The objectives of these studies were the identification of the receptor binding ligand(s) of P. berghei and the identification of proteins that can be used as vehicles to target P. falciparum var2CSA domains to the surface of P. berghei. By means of mass spectrometry, proteomes were generated of:
1. P. berghei iRBC membrane fractions (schisonts and trophosoites);
2. the surface of P. berghei iRBC membranes by treatment of intact schisonts with trypsin ('surface shaving');
3. specific exported protein complexes immunoprecipitated from P. berghei iRBC.

With all 3 approaches large protein sets were generated and iRBC surface candidates were selected by combining and comparing the data sets and by bio-informatic analysis of protein features such as the presence of transport elements. Selected surface candidates were further analysed by protein tagging to verify the presence on the schisont surface and gene disruption experiments to investigate possible involvement in parasite viability or sequestration.

Schizont membrane associated cytoadherance candidate (SMAC)

As has been described in the first report, one selected candidate from the iRBC membrane proteome (PBANKA_010060) was shown to be involved in CD36 sequestration of schisonts and was called SMAC. SMAC is a small protein (17 kDa) containing a signal sequence, a signal for export (PEXEL) and a C-terminal transmembrane region. Tagging of the SMAC protein with the red fluorescent reporter mCherry revealed localisation of SMAC in the erythrocyte cytoplasm of trophosoites and schizonts but not on the surface of the iRBC membrane, suggesting that SMAC itself is not the ligand for sequestration, but might be a protein that is involved in the transport or modification of the cytoadherence ligand. No clear SMAC orthologue could be identified in P. falciparum. The non-sequestering P. berghei SMAC parasite line was further implemented to permit the expression of P. falciparum receptor binding domains (e.g. PfEMP1 domains) to create a humanised mouse model for in vivo investigations of host-parasite interactions. Whether SMAC parasites are suitable to study cytoadherence in PAM has to be investigated. Studies in pregnant mice infected with the SMAC parasites showed that parasites were still accumulating in the placenta (personal communication L. Vieira de Moraes), indicating that this strain is not deficient in binding to CSA. This result would indicate that in P. berghei the mechanism of receptor binding is basically different from the PfEMP1 system in P. falciparum and that at least two independent ligand pathways exist for sequestration.

Pbfam1.1

To be able to more specifically identify surface proteins, we analysed the proteomes of so called 'surface shaved' iRBC of sequestering (wild type) and non-sequestering (SMAC, K173) P. berghei lines. In the data we found members of three P. berghei subtelomeric multigene families: bir, pbfam-1 and pbfam-3. Little is known about expression, subcellular location and function of these proteins but since they are part of a multigene family, a location at the surface of iRBC could be assumed. We C-terminally tagged several of these gene family members with mCherry or GFP and analysed their subcellular location and their potential use as transport vehicles for P. falciparum ligand domains. Fluorescence was detected in the erythrocyte cytoplasm of iRBC expressing tagged members of the bir gene family, the Pbfam-1 family and the Pbfam-3 family, indicating that members of these families are exported by the parasites. Only one of these tagged proteins, Pbfam1.1 showed an additional staining of the erythrocyte membrane and therefore was regarded as a key candidate for the implementation as transporter molecule for P. falciparum cytoadhesion domains to the erythrocyte surface. Pbfam1.1 is a 330 amino acid protein containing a signal peptide but no PEXEL (parasite export) motif or transmembrane region. Disruption of the Pbfam1.1 gene did not result in a CD36 non-sequestering phenotype suggesting that Pbfam1.1 is not the P. berghei ligand responsible for host-receptor binding. By immunofluorescence assays (IFA and FACS) on live, intact schizonts using antibodies to mCherry and Pbfam1.1 a strong surface staining was only observed of a small percentage (0.1-2 %) of iRBC and it could not be excluded that the staining was the result of a disturbed membrane integrity of these cells. Because it was still possible that Pbfam1.1 is exposed on the iRBC surface but that its presence is below the detection level of these assays, Pbfam1.1 was selected to investigate its ability to transport (functional) P. falciparum cytoadhesion domains to the membrane of P. berghei iRBC.

Expression of the Pbfam1.1::mCherry protein in the non-sequestering P. berghei SMAC strain revealed that Pbfam1.1 in this mutant line is also transported to the erythrocyte membrane. Therefore, Pbfam1.1 seems to be a suitable candidate to use as a tool to transport P. falciparum ligand domains to the iRBC membrane for in vivo cytoadherence studies. Interestingly, in the other available non-sequestering strain, K173, transport of Pbfam1.1 to the membrane was shown to be blocked.

In addition to the multi-gene family members, several single gene proteins were selected from the 'surface shaved' proteome for analysis by disruption and tagging. Although for some proteins export into the iRBC cytoplasm was observed, localisation in the erythrocyte membrane was not detected.

New surface candidate 1 (NSC1)

In an attempt to reduce the number of potential candidates obtained from the proteomes and to be able to focus more on proteins relevant to the project, we tried, by pull down experiments, to identify the proteins that are in complex with the two most interesting proteins we had identified thus far, SMAC and Pbfam1.1. With the pull down of SMAC we intended to identify the P. berghei receptor binding ligand or other proteins involved in sequestration. With the pull down of Pbfam1.1 we aimed at finding additional iRBC membrane proteins that could serve as a tool to traffic PfEMP1 domains to the erythrocyte surface. To do this we immunoprecipitated SMAC and Pbfam1.1 with associated proteins from schisont extracts of P. berghei transgenic lines expressing cMYC-tagged SMAC and Pbfam1.1 using antibodies to cMYC as well as antibodies specific to SMAC and Pbfam1.1. Pulled down proteins were analysed by mass spectrometry and once more a large set of P. berghei proteins was identified. By combining data of all different proteomes and by using bio-informatic analyses new candidate genes were selected for tagging and gene disruption. One of the interesting proteins was found in the pull downs of SMAC as well as Pbfam1.1. mCherry tagging revealed that this protein, like Pbfam1.1 was targeted to the membrane of the infected erythrocyte and therefore we called this protein NSC1. NSC1 is a 387 amino acid protein containing a signal peptide and a strong PEXEL motif, but no transmembrane region. The NSC1-gene is not a member of a multigene family and no orthologue is present in P. falciparum. Live IFA's on NSC1:mCherry expressing schisonts using mCherry antibodies showed clear fluorescence of the erythrocyte membrane for only a small percentage of schisonts (0.2-2 %), so like for Pbfam1.1 the precise surface exposure features of NSC1 is not clear. Given the similar localisation pattern of Pbfam1.1 and NSC1 and the fact they were coprecipitated in the pull down experiment, NSC1 and Pbfam1.1 might be co-localised. Disruption of the gene encoding NSC1 did not result in a CD36 non-sequestering phenotype so this protein is not considered as a host-receptor-binding ligand candidate. We will characterise NSC1 in more detail to obtain more insight into mechanisms underlying regulation of expression and erythrocyte surface exposure in wild type and non-sequestering P. berghei strains.

With NSC1 we have identified a second P. berghei protein that is located in the iRBC membrane and is a potential tool to fuse with receptor binding domains (e.g. var2CSA domains as established in WPs 1,3 and express in non-sequestering P. berghei strains to restore cytoadherence. In this way a humanised model for studying in vivo receptor-ligand interactions can be generated.

Targeting PfEMP1 domains to the surface of P. berghei iRBC

To traffic P. falciparum ligand domains to the erythrocyte surface, P. berghei expression constructs were made that encoded fusion proteins of Pbfam1.1 with PfEMP1 domains that have been shown (within and outside the consortium) to be involved in host receptor binding. Parasite lines were generated that express Pbfam1.1 fused C-terminally with DBL3-var2CSA_3D7 (CSA binding, from WP1), CIDR1fvo (CD36 binding, from WP1), DBL1-6var2CSA_3D7 (CSA binding, from WP1) and DBL1-6 var2CSA_fcr (CSA binding, from WP3). Assays were developed to determine whether Pbfam1.1 was able to functionally expose cytoadherent domains on the surface of iRBC transgenic P. berghei lines.

Pbfam1.1::CIDR1fvo

iRBC of mice infected with parasites expressing the Pbfam1.1::CIDR1fvo fusion were first tested for their ability to react with anti-CIDR1fvo monoclonal antibodies (from WP1) in live immunofluorescence assays. Like with the Pbfam1.1::mCherry parasites, the membranes of only a few iRBC were stained. To test whether the Pbfam1.1::CIDR1fvo fusion protein could function as a CD36 binding ligand, we expressed this protein in the non-sequestering SMAC parasite line that produces luciferase in the schizont stage. Above expectations, in vivo imaging of mice infected with these parasites showed a remarkable recovery of sequestration in comparison to the original SMAC parasite line. This indicates that the Pbfam1.1::CIDR1fvo fusion protein can be exposed on the surface of schizonts and is actually able to functionally bind to host receptor molecules. If additional experiments can confirm this 'recovery' of sequestering phenotype, this may create an in vivo screening system for testing inhibitors that block P. falciparum sequestration (drugs, specific antibodies) and studying host-parasite interactions.

Pbfam1.1:: DBL1-6 var2CSA

Parasites expressing the fusion product Pbfam1.1: DBL1-6var2CSA_3D7 (WP1) were analysed for expression and surface exposure of the DBL1-6 domains. A western blot of schizonts of the mutant line showed the expression of a >300kDa protein that was immunoreactive to a DBL1-6 specific antibody (from WP1). IFA's on live intact schizonts using the DBL1-6 antibody showed clear surface staining of a small percentage of infected cells. Subsequently we tested wild type and Pbfam1.1::DBL1-6var2CSA_3D7 expressing schizonts for their ability to bind to CSA and the proteoglycan decorin in vitro. No interaction of the wild type schizonts with these receptors was observed, showing that unselected P. berghei iRBC have no or only a weak endogenous affinity for CSA or decorin. DBL1-6 expressing parasites did not show binding to CSA but a clear interaction with immobilised decorin was detected. In addition, binding of the mutant schizonts to coated anti-DBL1-6 antibody was noticed, indicating that DBL1-6 was correctly folded and functionally exposed on the surface of Pbfam1.1::DBL1-6 var2CSA_3D7 expressing schisonts. It has to be demonstrated whether these parasites are able to bind in vitro to placenta sections or in vivo to placenta's in pregnant mice. Since it has been shown that the SMAC parasites were able to induce PAM in pregnant mice and to accumulate in the placenta's (personal communication L. Vieira de Moraes) it might be necessary to develop mutant P. berghei lines that do not exhibit intrinsic placental cytoadherence. Preliminary results indicated that the non-sequestering P. berghei K173 strain showed reduced parasite accumulation in mouse placenta. The use of this line for the expression and investigation of var2CSA binding domains will be investigated.

Potential impact:

Malaria has severe consequences for both public health and economic development in countries where the disease is endemic. Although estimates of global mortality is decreasing (around one million deaths per year, of which around 90 % occurs in sub-Saharan Africa), the impact of effective treatments for malaria is evident. In addition to high mortality, there are also severe consequences of morbidity arising from the 200 to 400 million clinical cases reported annually, which reduces the economic force and erodes the social fabric of these endemic regions. Young children, the most vulnerable section of the population, risk retardation in both their physical and mental development. The impact of effective treatments for malaria is thus evident. But Europe would also gain much in human, economic and political terms if the burden of this disease were lifted. While Europe has always been the leading force in basic malaria research, it has taken much effort to apply this success to the development of vaccine and anti-malarial strategies. The combat against malaria is a complex problem to which solutions require a concerted and long-term commitment. The European Commission's Fifth, Sixth and Seventh Framework Programmes have been important in bringing European applied malaria research to a world-class level and it is essential that the momentum and the benefits so far accrued from this investment are not lost.

Although the interaction between Plasmodium falciparum and host is complex, accumulated evidence strongly supports the idea that, in the case of PAM, PfEMP1 variants expressed by placental parasites are promising targets for developing new approaches to treat malaria. An effective strategy against PAM would indeed target a significant section of the vulnerable population in endemic regions of Sub-Saharan Africa and accordingly would have a significant impact on health and economy. Indeed, each year, malaria threatens 125 million pregnancies, and PAM is responsible for 100 000 to 250 000 infant deaths in sub-Saharan Africa. Moreover, simultaneous infection by HIV and P. falciparum, two plagues that often cohabit in these regions, enhances viral transmission from mother to foetus by mechanisms that are poorly understood. The overall objective of this project was to comprehend the functional and immunological characteristics of PfEMP1 variants expressed by placental parasites at the molecular level. Such knowledge is essential for providing a rational basis for accelerating vaccine and therapeutic developments to combat this form of malaria. Over three years, a major aim of this project was to provide knowledge to scientific groups working in the vaccine field. This project benefits from the synergy created by the parallel efforts made by all partners and thus make a significant impact on our basic knowledge of the molecular interaction between the human host and placental parasites.

The results generated during the PREMALSTRUCT project therefore have direct implications for the development of a malaria vaccine that would ease this burden of death and debilitation in sub-Saharan Africa and Asia. It is anticipated that PREMALSTRUCT partners will play in the near future an essential role in the development of the most promising PAM vaccine candidates.

List of websites: http://www.premalstruct.org

Final Report Summary - PREMALSTRUCT (Structural analysis of the CSA binding interactions involved during pregnancy associated malaria)

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