I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Daniel G. W.
Alanine
,
Doris
Quinkert
,
Rasika
Kumarasingha
,
Shahid
Mehmood
,
Francesca R.
Donnellan
,
Nana K.
Minkah
,
Bernadeta
Dadonaite
,
Ababacar
Diouf
,
Francis
Galaway
,
Sarah E.
Silk
,
Abhishek
Jamwal
,
Jennifer M.
Marshall
,
Kazutoyo
Miura
,
Lander
Foquet
,
Sean C.
Elias
,
Geneviève M.
Labbé
,
Alexander D.
Douglas
,
Jing
Jin
,
Ruth O.
Payne
,
Joseph J.
Illingworth
,
David J.
Pattinson
,
David
Pulido
,
Barnabas G.
Williams
,
Willem A.
De Jongh
,
Gavin J.
Wright
,
Stefan H. I.
Kappe
,
Carol V.
Robinson
,
Carole A.
Long
,
Brendan S.
Crabb
,
Paul R.
Gilson
,
Matthew
Higgins
,
Simon J.
Draper
Diamond Proposal Number(s):
[18069]
Open Access
Abstract: The Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) is the leading target for next-generation vaccines against the disease-causing blood-stage of malaria. However, little is known about how human antibodies confer functional immunity against this antigen. We isolated a panel of human monoclonal antibodies (mAbs) against PfRH5 from peripheral blood B cells from vaccinees in the first clinical trial of a PfRH5-based vaccine. We identified a subset of mAbs with neutralizing activity that bind to three distinct sites and another subset of mAbs that are non-functional, or even antagonistic to neutralizing antibodies. We also identify the epitope of a novel group of non-neutralizing antibodies that significantly reduce the speed of red blood cell invasion by the merozoite, thereby potentiating the effect of all neutralizing PfRH5 antibodies as well as synergizing with antibodies targeting other malaria invasion proteins. Our results provide a roadmap for structure-guided vaccine development to maximize antibody efficacy against blood-stage malaria.
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Jun 2019
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I03-Macromolecular Crystallography
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Thomas. A.
Rawlinson
,
Natalie M.
Barber
,
Franziska
Mohring
,
Jee Sun
Cho
,
Varakorn
Kosaisavee
,
Samuel F.
Gerard
,
Daniel G. W.
Alanine
,
Geneviève M.
Labbé
,
Sean C.
Elias
,
Sarah E.
Silk
,
Doris
Quinkert
,
Jing
Jin
,
Jennifer M.
Marshall
,
Ruth O.
Payne
,
Angela M.
Minassian
,
Bruce
Russell
,
Laurent
Rénia
,
François H.
Nosten
,
Robert W.
Moon
,
Matthew K.
Higgins
,
Simon J.
Draper
Diamond Proposal Number(s):
[18069]
Abstract: The most widespread form of malaria is caused by Plasmodium vivax. To replicate, this parasite must invade immature red blood cells through a process requiring interaction of the P. vivax Duffy binding protein (PvDBP) with its human receptor, the Duffy antigen receptor for chemokines. Naturally acquired antibodies that inhibit this interaction associate with clinical immunity, suggesting PvDBP as a leading candidate for inclusion in a vaccine to prevent malaria due to P. vivax. Here, we isolated a panel of monoclonal antibodies from human volunteers immunized in a clinical vaccine trial of PvDBP. We screened their ability to prevent PvDBP from binding to the Duffy antigen receptor for chemokines, and their capacity to block red blood cell invasion by a transgenic Plasmodium knowlesi parasite genetically modified to express PvDBP and to prevent reticulocyte invasion by multiple clinical isolates of P. vivax. This identified a broadly neutralizing human monoclonal antibody that inhibited invasion of all tested strains of P. vivax. Finally, we determined the structure of a complex of this antibody bound to PvDBP, indicating the molecular basis for inhibition. These findings will guide future vaccine design strategies and open up possibilities for testing the prophylactic use of such an antibody.
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May 2019
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I03-Macromolecular Crystallography
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Frank
Lennartz
,
Florian
Brod
,
Rebecca
Dabbs
,
Kazutoyo
Miura
,
David
Mekhaiel
,
Arianna
Marini
,
Matthijs
Jore
,
Max M.
Søgaard
,
Thomas
Jørgensen
,
Willem A.
De Jongh
,
Robert W.
Sauerwein
,
Carole A.
Long
,
Sumi
Biswas
,
Matthew
Higgins
Diamond Proposal Number(s):
[18069]
Open Access
Abstract: The quest to develop an effective malaria vaccine remains a major priority in the fight against global infectious disease. An approach with great potential is a transmission-blocking vaccine which induces antibodies that prevent establishment of a productive infection in mosquitos that feed on infected humans, thereby stopping the transmission cycle. One of the most promising targets for such a vaccine is the gamete surface protein, Pfs48/45. Here we establish a system for production of full-length Pfs48/45 and use this to raise a panel of monoclonal antibodies. We map the binding regions of these antibodies on Pfs48/45 and correlate the location of their epitopes with their transmission-blocking activity. Finally, we present the structure of the C-terminal domain of Pfs48/45 bound to the most potent transmission-blocking antibody, and provide key molecular information for future structure-guided immunogen design.
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Sep 2018
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[12346]
Abstract: Only two trypanosome subspecies are able to cause human African trypanosomiasis. To establish an infection in human blood, they must overcome the innate immune system by resisting the toxic effects of trypanolytic factor 1 and trypanolytic factor 2 (refs. 1,2). These lipoprotein complexes contain an active, pore-forming component, apolipoprotein L1 (ApoL1), that causes trypanosome cell death3. One of the two human-infective subspecies, Trypanosoma brucei rhodesiense, differs from non-infective trypanosomes solely by the presence of the serum resistance-associated protein, which binds directly to ApoL1 and blocks its pore-forming capacity3,4,5. Since this interaction is the single critical event that renders T. b. rhodesiense human- infective, detailed structural information that allows identification of binding determinants is crucial to understand immune escape by the parasite. Here, we present the structure of serum resistance-associated protein and reveal the adaptations that occurred as it diverged from other trypanosome surface molecules to neutralize ApoL1. We also present our mapping of residues important for ApoL1 binding, giving molecular insight into this interaction at the heart of human sleeping sickness.
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Jan 2018
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[12346]
Open Access
Abstract: Antibodies are critical components of the human adaptive immune system, providing versatile scaffolds to display diverse antigen-binding surfaces. Nevertheless, most antibodies have similar architectures, with the variable immunoglobulin domains of the heavy and light chain each providing three hypervariable loops, which are varied to generate diversity. The recent identification of a novel class of antibody in humans from malaria endemic regions of Africa was therefore surprising as one hypervariable loop contains the entire collagen-binding domain of human LAIR1. Here, we present the structure of the Fab fragment of such an antibody. We show that its antigen-binding site has adopted an architecture that positions LAIR1, while itself being occluded. This therefore represents a novel means of antigen recognition, in which the Fab fragment of an antibody acts as an adaptor, linking a human protein insert with antigen-binding potential to the constant antibody regions which mediate immune cell recruitment.
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May 2017
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I02-Macromolecular Crystallography
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Frank
Lennartz
,
Yvonne
Adams
,
Anja
Bengtsson
,
Rebecca W.
Olsen
,
Louise
Turner
,
Nicaise T.
Ndam
,
Gertrude
Ecklu-mensah
,
Azizath
Moussiliou
,
Michael F.
Ofori
,
Benoit
Gamain
,
John P.
Lusingu
,
Jens E. V.
Petersen
,
Christian W.
Wang
,
Sofia
Nunes-silva
,
Jakob S.
Jespersen
,
Clinton K. Y.
Lau
,
Thor G.
Theander
,
Thomas
Lavstsen
,
Lars
Hviid
,
Matthew K.
Higgins
,
Anja T. R.
Jensen
Open Access
Abstract: Cerebral malaria is a deadly outcome of infection by Plasmodium falciparum, occurring when parasite-infected erythrocytes accumulate in the brain. These erythrocytes display parasite proteins of the PfEMP1 family that bind various endothelial receptors. Despite the importance of cerebral malaria, a binding phenotype linked to its symptoms has not been identified. Here, we used structural biology to determine how a group of PfEMP1 proteins interacts with intercellular adhesion molecule 1 (ICAM-1), allowing us to predict binders from a specific sequence motif alone. Analysis of multiple Plasmodium falciparum genomes showed that ICAM-1-binding PfEMP1s also interact with endothelial protein C receptor (EPCR), allowing infected erythrocytes to synergistically bind both receptors. Expression of these PfEMP1s, predicted to bind both ICAM-1 and EPCR, is associated with increased risk of developing cerebral malaria. This study therefore reveals an important PfEMP1-binding phenotype that could be targeted as part of a strategy to prevent cerebral malaria.
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Mar 2017
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[12346]
Open Access
Abstract: Many promising vaccine candidates from pathogenic viruses, bacteria, and parasites are unstable and cannot be produced cheaply for clinical use. For instance, Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) is essential for erythrocyte invasion, is highly conserved among field isolates, and elicits antibodies that neutralize in vitro and protect in an animal model, making it a leading malaria vaccine candidate. However, functional RH5 is only expressible in eukaryotic systems and exhibits moderate temperature tolerance, limiting its usefulness in hot and low-income countries where malaria prevails. Current approaches to immunogen stabilization involve iterative application of rational or semirational design, random mutagenesis, and biochemical characterization. Typically, each round of optimization yields minor improvement in stability, and multiple rounds are required. In contrast, we developed a one-step design strategy using phylogenetic analysis and Rosetta atomistic calculations to design PfRH5 variants with improved packing and surface polarity. To demonstrate the robustness of this approach, we tested three PfRH5 designs, all of which showed improved stability relative to wild type. The best, bearing 18 mutations relative to PfRH5, expressed in a folded form in bacteria at >1 mg of protein per L of culture, and had 10–15 °C higher thermal tolerance than wild type, while also retaining ligand binding and immunogenic properties indistinguishable from wild type, proving its value as an immunogen for a future generation of vaccines against the malaria blood stage. We envision that this efficient computational stability design methodology will also be used to enhance the biophysical properties of other recalcitrant vaccine candidates from emerging pathogens.
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Jan 2017
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[9306]
Open Access
Abstract: CD36 is a scavenger receptor involved in fatty acid metabolism, innate immunity and angiogenesis. It interacts with lipoprotein particles and facilitates uptake of long chain fatty acids. It is also the most common target of the PfEMP1 proteins of the malaria parasite, Plasmodium falciparum, tethering parasite-infected erythrocytes to endothelial receptors. This prevents their destruction by splenic clearance and allows increased parasitaemia. Here we describe the structure of CD36 in complex with long chain fatty acids and a CD36-binding PfEMP1 protein domain. A conserved hydrophobic pocket allows the hugely diverse PfEMP1 protein family to bind to a conserved phenylalanine residue at the membrane distal tip of CD36. This phenylalanine is also required for CD36 to interact with lipoprotein particles. By targeting a site on CD36 that is required for its physiological function, PfEMP1 proteins maintain the ability to tether to the endothelium and avoid splenic clearance.
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Sep 2016
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[9306]
Open Access
Abstract: The haptoglobin-haemoglobin receptor of the African trypanosome species, Trypanosoma brucei, is expressed when the parasite is in the bloodstream of the mammalian host, allowing it to acquire haem through the uptake of haptoglobin-haemoglobin complexes. Here we show that in Trypanosoma congolense this receptor is instead expressed in the epimastigote developmental stage that occurs in the tsetse fly, where it acts as a haemoglobin receptor. We also present the structure of the T. congolense receptor in complex with haemoglobin. This allows us to propose an evolutionary history for this receptor, charting the structural and cellular changes that took place as it adapted from a role in the insect to a new role in the mammalian host.
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Apr 2016
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Clinton K. Y.
Lau
,
Louise
Turner
,
Jakob S.
Jespersen
,
Edward D.
Lowe
,
Bent
Petersen
,
Christian W.
Wang
,
Jens E. V.
Petersen
,
John
Lusingu
,
Thor G.
Theander
,
Thomas
Lavstsen
,
Matthew K.
Higgins
Open Access
Abstract: The PfEMP1 family of surface proteins is central for Plasmodium falciparum virulence and must retain the ability to bind to host receptors while also diversifying to aid immune evasion. The interaction between CIDRα1 domains of PfEMP1 and endothelial protein C receptor (EPCR) is associated with severe childhood malaria. We combine crystal structures of CIDRα1:EPCR complexes with analysis of 885 CIDRα1 sequences, showing that the EPCR-binding surfaces of CIDRα1 domains are conserved in shape and bonding potential, despite dramatic sequence diversity. Additionally, these domains mimic features of the natural EPCR ligand and can block this ligand interaction. Using peptides corresponding to the EPCR-binding region, antibodies can be purified from individuals in malaria-endemic regions that block EPCR binding of diverse CIDRα1 variants. This highlights the extent to which such a surface protein family can diversify while maintaining ligand-binding capacity and identifies features that should be mimicked in immunogens to prevent EPCR binding.
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Jan 2015
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