RESUMEN
There is an urgent need for improved malaria vaccine immunogens. Invasion of erythrocytes by Plasmodium falciparum is essential for its life cycle, preceding symptoms of disease and parasite transmission. Antibodies which target PfRH5 are highly effective at preventing erythrocyte invasion and the most potent growth-inhibitory antibodies bind a single epitope. Here we use structure-guided approaches to design a small synthetic immunogen, RH5-34EM which recapitulates this epitope. Structural biology and biophysics demonstrate that RH5-34EM is correctly folded and binds neutralising monoclonal antibodies with nanomolar affinity. In immunised rats, RH5-34EM induces PfRH5-targeting antibodies that inhibit parasite growth. While PfRH5-specific antibodies were induced at a lower concentration by RH5-34EM than by PfRH5, RH5-34EM induced antibodies that were a thousand-fold more growth-inhibitory as a factor of PfRH5-specific antibody concentration. Finally, we show that priming with RH5-34EM and boosting with PfRH5 achieves the best balance between antibody quality and quantity and induces the most effective growth-inhibitory response. This rationally designed vaccine immunogen is now available for use as part of future malaria vaccines, alone or in combination with other immunogens.
RESUMEN
Plasmodium falciparum reticulocyte-binding protein homolog 5 (RH5) is the most advanced blood-stage malaria vaccine candidate and is being evaluated for efficacy in endemic regions, emphasizing the need to study the underlying antibody response to RH5 during natural infection, which could augment or counteract responses to vaccination. Here, we found that RH5-reactive B cells were rare, and circulating immunoglobulin G (IgG) responses to RH5 were short-lived in malaria-exposed Malian individuals, despite repeated infections over multiple years. RH5-specific monoclonal antibodies isolated from eight malaria-exposed individuals mostly targeted non-neutralizing epitopes, in contrast to antibodies isolated from five RH5-vaccinated, malaria-naive UK individuals. However, MAD8-151 and MAD8-502, isolated from two malaria-exposed Malian individuals, were among the most potent neutralizers out of 186 antibodies from both cohorts and targeted the same epitopes as the most potent vaccine-induced antibodies. These results suggest that natural malaria infection may boost RH5-vaccine-induced responses and provide a clear strategy for the development of next-generation RH5 vaccines.
Asunto(s)
Anticuerpos Neutralizantes , Anticuerpos Antiprotozoarios , Antígenos de Protozoos , Vacunas contra la Malaria , Malaria Falciparum , Plasmodium falciparum , Humanos , Anticuerpos Neutralizantes/inmunología , Plasmodium falciparum/inmunología , Malaria Falciparum/inmunología , Malaria Falciparum/prevención & control , Malaria Falciparum/parasitología , Vacunas contra la Malaria/inmunología , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulina G/sangre , Proteínas Protozoarias/inmunología , Anticuerpos Monoclonales/inmunología , Adulto , Linfocitos B/inmunología , Epítopos/inmunología , Femenino , Malí , Proteínas Portadoras/inmunología , Masculino , AdolescenteRESUMEN
IL-33 plays a significant role in inflammation, allergy, and host defence against parasitic helminths. The model gastrointestinal nematode Heligmosomoides polygyrus bakeri secretes the Alarmin Release Inhibitor HpARI2, an effector protein that suppresses protective immune responses and asthma in its host by inhibiting IL-33 signalling. Here we reveal the structure of HpARI2 bound to mouse IL-33. HpARI2 contains three CCP-like domains, and we show that it contacts IL-33 primarily through the second and third of these. A large loop which emerges from CCP3 directly contacts IL-33 and structural comparison shows that this overlaps with the binding site on IL-33 for its receptor, ST2, preventing formation of a signalling complex. Truncations of HpARI2 which lack the large loop from CCP3 are not able to block IL-33-mediated signalling in a cell-based assay and in an in vivo female mouse model of asthma. This shows that direct competition between HpARI2 and ST2 is responsible for suppression of IL-33-dependent responses.
Asunto(s)
Asma , Proteínas del Helminto , Proteína 1 Similar al Receptor de Interleucina-1 , Interleucina-33 , Nematospiroides dubius , Animales , Interleucina-33/metabolismo , Interleucina-33/química , Nematospiroides dubius/inmunología , Proteínas del Helminto/metabolismo , Proteínas del Helminto/química , Proteínas del Helminto/inmunología , Ratones , Femenino , Proteína 1 Similar al Receptor de Interleucina-1/metabolismo , Asma/inmunología , Asma/metabolismo , Humanos , Transducción de Señal , Infecciones por Strongylida/inmunología , Infecciones por Strongylida/parasitología , Infecciones por Strongylida/metabolismo , Unión Proteica , Modelos Animales de Enfermedad , Sitios de Unión , Ratones Endogámicos BALB C , Ratones Endogámicos C57BLRESUMEN
Reticulocyte-binding protein homologue 5 (RH5), a leading blood-stage Plasmodium falciparum malaria vaccine target, interacts with cysteine-rich protective antigen (CyRPA) and RH5-interacting protein (RIPR) to form an essential heterotrimeric "RCR-complex". We investigate whether RCR-complex vaccination can improve upon RH5 alone. Using monoclonal antibodies (mAbs) we show that parasite growth-inhibitory epitopes on each antigen are surface-exposed on the RCR-complex and that mAb pairs targeting different antigens can function additively or synergistically. However, immunisation of female rats with the RCR-complex fails to outperform RH5 alone due to immuno-dominance of RIPR coupled with inferior potency of anti-RIPR polyclonal IgG. We identify that all growth-inhibitory antibody epitopes of RIPR cluster within the C-terminal EGF-like domains and that a fusion of these domains to CyRPA, called "R78C", combined with RH5, improves the level of in vitro parasite growth inhibition compared to RH5 alone. These preclinical data justify the advancement of the RH5.1 + R78C/Matrix-M™ vaccine candidate to Phase 1 clinical trial.
Asunto(s)
Anticuerpos Monoclonales , Anticuerpos Antiprotozoarios , Antígenos de Protozoos , Vacunas contra la Malaria , Malaria Falciparum , Plasmodium falciparum , Proteínas Protozoarias , Vacunas contra la Malaria/inmunología , Vacunas contra la Malaria/administración & dosificación , Animales , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Femenino , Malaria Falciparum/prevención & control , Malaria Falciparum/inmunología , Malaria Falciparum/parasitología , Antígenos de Protozoos/inmunología , Ratas , Anticuerpos Antiprotozoarios/inmunología , Anticuerpos Monoclonales/inmunología , Humanos , Epítopos/inmunología , Proteínas Portadoras/inmunología , Proteínas Portadoras/metabolismoRESUMEN
African trypanosomes replicate within infected mammals where they are exposed to the complement system. This system centres around complement C3, which is present in a soluble form in serum but becomes covalently deposited onto the surfaces of pathogens after proteolytic cleavage to C3b. Membrane-associated C3b triggers different complement-mediated effectors which promote pathogen clearance. To counter complement-mediated clearance, African trypanosomes have a cell surface receptor, ISG65, which binds to C3b and which decreases the rate of trypanosome clearance in an infection model. However, the mechanism by which ISG65 reduces C3b function has not been determined. We reveal through cryogenic electron microscopy that ISG65 has two distinct binding sites for C3b, only one of which is available in C3 and C3d. We show that ISG65 does not block the formation of C3b or the function of the C3 convertase which catalyses the surface deposition of C3b. However, we show that ISG65 forms a specific conjugate with C3b, perhaps acting as a decoy. ISG65 also occludes the binding sites for complement receptors 2 and 3, which may disrupt recruitment of immune cells, including B cells, phagocytes, and granulocytes. This suggests that ISG65 protects trypanosomes by combining multiple approaches to dampen the complement cascade.
Asunto(s)
Complemento C3b , Complemento C3b/metabolismo , Humanos , Unión Proteica , Trypanosoma brucei brucei/inmunología , Trypanosoma brucei brucei/metabolismo , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/inmunología , Microscopía por Crioelectrón , Sitios de Unión , Complemento C3/metabolismo , Complemento C3/inmunologíaRESUMEN
HpARI is an immunomodulatory protein secreted by the intestinal nematode Heligmosomoides polygyrus bakeri, which binds and blocks IL-33. Here, we find that the H. polygyrus bakeri genome contains three HpARI family members and that these have different effects on IL-33-dependent responses in vitro and in vivo, with HpARI1+2 suppressing and HpARI3 amplifying these responses. All HpARIs have sub-nanomolar affinity for mouse IL-33; however, HpARI3 does not block IL-33-ST2 interactions. Instead, HpARI3 stabilizes IL-33, increasing the half-life of the cytokine and amplifying responses to it in vivo. Together, these data show that H. polygyrus bakeri secretes a family of HpARI proteins with both overlapping and distinct functions, comprising a complex immunomodulatory arsenal of host-targeted proteins.
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Nematospiroides dubius , Infecciones por Strongylida , Ratones , Animales , Interleucina-33/genética , Citocinas , Inmunomodulación , InmunidadRESUMEN
The symptoms of malaria occur during the blood stage of infection, when parasites invade and replicate within human erythrocytes. The PfPCRCR complex1, containing PfRH5 (refs. 2,3), PfCyRPA, PfRIPR, PfCSS and PfPTRAMP, is essential for erythrocyte invasion by the deadliest human malaria parasite, Plasmodium falciparum. Invasion can be prevented by antibodies3-6 or nanobodies1 against each of these conserved proteins, making them the leading blood-stage malaria vaccine candidates. However, little is known about how PfPCRCR functions during invasion. Here we present the structure of the PfRCR complex7,8, containing PfRH5, PfCyRPA and PfRIPR, determined by cryogenic-electron microscopy. We test the hypothesis that PfRH5 opens to insert into the membrane9, instead showing that a rigid, disulfide-locked PfRH5 can mediate efficient erythrocyte invasion. We show, through modelling and an erythrocyte-binding assay, that PfCyRPA-binding antibodies5 neutralize invasion through a steric mechanism. We determine the structure of PfRIPR, showing that it consists of an ordered, multidomain core flexibly linked to an elongated tail. We also show that the elongated tail of PfRIPR, which is the target of growth-neutralizing antibodies6, binds to the PfCSS-PfPTRAMP complex on the parasite membrane. A modular PfRIPR is therefore linked to the merozoite membrane through an elongated tail, and its structured core presents PfCyRPA and PfRH5 to interact with erythrocyte receptors. This provides fresh insight into the molecular mechanism of erythrocyte invasion and opens the way to new approaches in rational vaccine design.
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Eritrocitos , Malaria Falciparum , Complejos Multiproteicos , Parásitos , Plasmodium falciparum , Proteínas Protozoarias , Animales , Humanos , Anticuerpos Neutralizantes/inmunología , Antígenos de Protozoos/química , Antígenos de Protozoos/inmunología , Microscopía por Crioelectrón , Disulfuros/química , Disulfuros/metabolismo , Eritrocitos/metabolismo , Eritrocitos/parasitología , Vacunas contra la Malaria/inmunología , Malaria Falciparum/inmunología , Malaria Falciparum/metabolismo , Malaria Falciparum/parasitología , Malaria Falciparum/patología , Merozoítos/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/inmunología , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/ultraestructura , Parásitos/metabolismo , Parásitos/patogenicidad , Plasmodium falciparum/metabolismo , Plasmodium falciparum/patogenicidad , Proteínas Protozoarias/química , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/ultraestructuraRESUMEN
Basigin is an essential host receptor for invasion of Plasmodium falciparum into human erythrocytes, interacting with parasite surface protein PfRH5. PfRH5 is a leading blood-stage malaria vaccine candidate and a target of growth-inhibitory antibodies. Here, we show that erythrocyte basigin is exclusively found in one of two macromolecular complexes, bound either to plasma membrane Ca2+-ATPase 1/4 (PMCA1/4) or to monocarboxylate transporter 1 (MCT1). PfRH5 binds to each of these complexes with a higher affinity than to isolated basigin ectodomain, making it likely that these are the physiological targets of PfRH5. PMCA-mediated Ca2+ export is not affected by PfRH5, making it unlikely that this is the mechanism underlying changes in calcium flux at the interface between an erythrocyte and the invading parasite. However, our studies rationalise the function of the most effective growth-inhibitory antibodies targeting PfRH5. While these antibodies do not reduce the binding of PfRH5 to monomeric basigin, they do reduce its binding to basigin-PMCA and basigin-MCT complexes. This indicates that the most effective PfRH5-targeting antibodies inhibit growth by sterically blocking the essential interaction of PfRH5 with basigin in its physiological context.
Asunto(s)
Malaria Falciparum , Plasmodium falciparum , Humanos , Plasmodium falciparum/fisiología , Basigina , Eritrocitos/parasitología , Anticuerpos Neutralizantes , Malaria Falciparum/parasitología , Proteínas Protozoarias/metabolismo , Unión Proteica , Antígenos de ProtozoosRESUMEN
African trypanosomes show a remarkable ability to survive as extracellular parasites in the blood and tissue spaces of an infected mammal. Throughout the infection they are exposed to the molecules and cells of the immune system, including complement. In this opinion piece, we review decades-worth of evidence about how complement affects African trypanosomes. We highlight the discovery of a trypanosome receptor for complement C3 and we critically assess three recent studies which attempt to provide a structural and mechanistic view of how this receptor helps trypanosomes to survive in the presence of complement.
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Trypanosoma , Tripanosomiasis Africana , Animales , Tripanosomiasis Africana/parasitología , MamíferosRESUMEN
The symptoms of malaria occur during the blood stage of infection, when the parasite replicates within human red blood cells. The human malaria parasite, Plasmodium vivax, selectively invades reticulocytes in a process which requires an interaction between the ectodomain of the human DARC receptor and the Plasmodium vivax Duffy-binding protein, PvDBP. Previous studies have revealed that a small helical peptide from DARC binds to region II of PvDBP (PvDBP-RII). However, it is also known that sulphation of tyrosine residues on DARC affects its binding to PvDBP and these residues were not observed in previous structures. We therefore present the structure of PvDBP-RII bound to sulphated DARC peptide, showing that a sulphate on tyrosine 41 binds to a charged pocket on PvDBP-RII. We use molecular dynamics simulations, affinity measurements and growth-inhibition experiments in parasites to confirm the importance of this interaction. We also reveal the epitope for vaccine-elicited growth-inhibitory antibody DB1. This provides a complete understanding of the binding of PvDBP-RII to DARC and will guide the design of vaccines and therapeutics to target this essential interaction.
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Sistema del Grupo Sanguíneo Duffy , Malaria Vivax , Plasmodium vivax , Humanos , Antígenos de Protozoos , Eritrocitos/parasitología , Malaria Vivax/parasitología , Plasmodium vivax/metabolismo , Proteínas Protozoarias/metabolismo , Reticulocitos/metabolismo , Tirosina/metabolismoRESUMEN
An effective malaria vaccine remains a global health priority and vaccine immunogens which prevent transmission of the parasite will have important roles in multi-component vaccines. One of the most promising candidates for inclusion in a transmission-blocking malaria vaccine is the gamete surface protein Pfs48/45, which is essential for development of the parasite in the mosquito midgut. Indeed, antibodies which bind Pfs48/45 can prevent transmission if ingested with the parasite as part of the mosquito bloodmeal. Here we present the structure of full-length Pfs48/45, showing its three domains to form a dynamic, planar, triangular arrangement. We reveal where transmission-blocking and non-blocking antibodies bind on Pfs48/45. Finally, we demonstrate that antibodies which bind across this molecule can be transmission-blocking. These studies will guide the development of future Pfs48/45-based vaccine immunogens.
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Vacunas contra la Malaria , Malaria Falciparum , Animales , Anticuerpos Bloqueadores , Anticuerpos Antiprotozoarios , Malaria Falciparum/parasitología , Proteínas de la Membrana , Plasmodium falciparum , Proteínas Protozoarias/químicaRESUMEN
African trypanosomes are extracellular pathogens of mammals and are exposed to the adaptive and innate immune systems. Trypanosomes evade the adaptive immune response through antigenic variation, but little is known about how they interact with components of the innate immune response, including complement. Here we demonstrate that an invariant surface glycoprotein, ISG65, is a receptor for complement component 3 (C3). We show how ISG65 binds to the thioester domain of C3b. We also show that C3 contributes to control of trypanosomes during early infection in a mouse model and provide evidence that ISG65 is involved in reducing trypanosome susceptibility to C3-mediated clearance. Deposition of C3b on pathogen surfaces, such as trypanosomes, is a central point in activation of the complement system. In ISG65, trypanosomes have evolved a C3 receptor which diminishes the downstream effects of C3 deposition on the control of infection.
Asunto(s)
Glicoproteínas de Membrana/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei , Trypanosoma , Animales , Complemento C3 , Antígeno de Macrófago-1 , Mamíferos/metabolismo , Ratones , Trypanosoma/fisiología , Trypanosoma brucei brucei/metabolismoRESUMEN
Plasmodium multigene families are thought to play important roles in the pathogenesis of malaria. Plasmodium interspersed repeat (pir) genes comprise the largest multigene family in many Plasmodium species. However, their expression pattern and localisation remain to be elucidated. Understanding protein subcellular localisation is fundamental to reveal the functional importance and cell-cell interactions of the PIR proteins. Here, we use the rodent malaria parasite, Plasmodium chabaudi chabaudi, as a model to investigate the localisation pattern of this gene family. We found that most PIR proteins are co-expressed in clusters during acute and chronic infection; members of the S7 clade are predominantly expressed during the acute-phase, whereas members of the L1 clade dominate the chronic-phase of infection. Using peptide antisera specific for S7 or L1 PIRS, we show that these PIRs have different localisations within the infected red blood cells. S7 PIRs are exported into the infected red blood cell cytoplasm where they are co-localised with parasite-induced host cell modifications termed Maurer's clefts, whereas L1 PIRs are localised on or close to the parasitophorous vacuolar membrane. This localisation pattern changes following mosquito transmission and during progression from acute- to chronic-phase of infection. The presence of PIRs in Maurer's clefts, as seen for Plasmodium falciparum RIFIN and STEVOR proteins, might suggest trafficking of the PIRs on the surface of the infected erythrocytes. However, neither S7 nor L1 PIR proteins detected by the peptide antisera are localised on the surface of infected red blood cells, suggesting that they are unlikely to be targets of surface variant-specific antibodies or to be directly involved in adhesion of infected red blood cells to host cells, as described for Plasmodium falciparum VAR proteins. The differences in subcellular localisation of the two major clades of Plasmodium chabaudi PIRs across the blood cycle, and the apparent lack of expression on the red cell surface strongly suggest that the function(s) of this gene family may differ from those of other multigene families of Plasmodium, such as the var genes of Plasmodium falciparum.
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Malaria , Plasmodium , Animales , Eritrocitos , Sueros Inmunes/metabolismo , Plasmodium falciparum/genéticaRESUMEN
A detailed understanding of the interaction between the highly variant Plasmodium falciparum erythrocyte membrane proteins 1 (PfEMP1) and their human binding partners is essential to explain their roles in disease development in malaria, as well as to understand how antibodies can inhibit these interactions and how the parasite manages to evade such an immune response. This chapter focuses on using surface plasmon resonance (SPR) as a reproducible, high-throughput method to quantitatively characterize these interactions. We describe how to utilize protein A or A/G and streptavidin for protein immobilization on SPR sensor chips and provide instructions on how to biotinylate proteins for this purpose and how to use SPR for binding competition assays. Since these experiments rely on recombinant proteins, we also present a method to verify their structural integrity using circular dichroism spectroscopy.
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Malaria Falciparum , Plasmodium falciparum , Anticuerpos Antiprotozoarios , Proteínas Portadoras/metabolismo , Eritrocitos/metabolismo , Humanos , Malaria Falciparum/parasitología , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes/metabolismo , Resonancia por Plasmón de SuperficieRESUMEN
Understanding mechanisms of antibody synergy is important for vaccine design and antibody cocktail development. Examples of synergy between antibodies are well-documented, but the mechanisms underlying these relationships often remain poorly understood. The leading blood-stage malaria vaccine candidate, CyRPA, is essential for invasion of Plasmodium falciparum into human erythrocytes. Here we present a panel of anti-CyRPA monoclonal antibodies that strongly inhibit parasite growth in in vitro assays. Structural studies show that growth-inhibitory antibodies bind epitopes on a single face of CyRPA. We also show that pairs of non-competing inhibitory antibodies have strongly synergistic growth-inhibitory activity. These antibodies bind to neighbouring epitopes on CyRPA and form lateral, heterotypic interactions which slow antibody dissociation. We predict that such heterotypic interactions will be a feature of many immune responses. Immunogens which elicit such synergistic antibody mixtures could increase the potency of vaccine-elicited responses to provide robust and long-lived immunity against challenging disease targets.
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Anticuerpos Monoclonales/inmunología , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/inmunología , Vacunas contra la Malaria/inmunología , Malaria Falciparum/prevención & control , Proteínas Protozoarias/inmunología , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Monoclonales/metabolismo , Anticuerpos Antiprotozoarios/aislamiento & purificación , Anticuerpos Antiprotozoarios/metabolismo , Antígenos de Protozoos/genética , Antígenos de Protozoos/aislamiento & purificación , Antígenos de Protozoos/metabolismo , Línea Celular , Drosophila melanogaster , Epítopos/inmunología , Humanos , Inmunogenicidad Vacunal , Vacunas contra la Malaria/uso terapéutico , Malaria Falciparum/inmunología , Malaria Falciparum/parasitología , Plasmodium falciparum/inmunología , Proteínas Protozoarias/genética , Proteínas Protozoarias/aislamiento & purificación , Proteínas Protozoarias/metabolismo , Desarrollo de VacunasRESUMEN
African trypanosomes cause diseases of humans and their livestock. To date, a much-desired vaccine has been elusive, due in part to the immune evasion mechanisms of these cunning parasites. However, Autheman et al. have used a bold, high-throughput screen to provide hope that vaccines may be on the way.
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Vacunas Antiprotozoos , Tripanosomiasis Africana , Animales , Interacciones Huésped-Parásitos/inmunología , Humanos , Evasión Inmune/inmunología , Trypanosoma/inmunología , Tripanosomiasis Africana/prevención & controlRESUMEN
The announcement of the outstanding performance of AlphaFold 2 in the CASP 14 protein structure prediction competition came at the end of a long year defined by the COVID-19 pandemic. With an infectious organism dominating the world stage, the developers of Alphafold 2 were keen to play their part, accurately predicting novel structures of two proteins from SARS-CoV-2. In their blog post of December 2020, they highlighted this contribution, writing "we've also seen signs that protein structure prediction could be useful in future pandemic response efforts". So, what role does structural biology play in guiding vaccine immunogen design and what might be the contribution of AlphaFold 2?
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Pandemias/prevención & control , Programas Informáticos , Vacunas/química , Anticuerpos Neutralizantes/inmunología , Diseño de Fármacos , Epítopos/química , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/inmunología , Conformación ProteicaRESUMEN
In this issue, Adams et al. (2021. J. Exp. Med. https://doi.org/10.1084/jem.20201266) show that red blood cells infected with strains of Plasmodium falciparum, which are commonly found in cerebral malaria patients, are specifically internalized by brain endothelial cells, perhaps contributing to the symptoms of the disease.
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Malaria , Parásitos , Animales , Encéfalo , Células Endoteliales , Eritrocitos , Humanos , Plasmodium falciparumRESUMEN
Structure-guided vaccine design provides a route to elicit a focused immune response against the most functionally important regions of a pathogen surface. This can be achieved by identifying epitopes for neutralizing antibodies through structural methods and recapitulating these epitopes by grafting their core structural features onto smaller scaffolds. In this study, we conducted a modified version of this protocol. We focused on the PfEMP1 protein family found on the surfaces of erythrocytes infected with Plasmodium falciparum A subset of PfEMP1 proteins bind to endothelial protein C receptor (EPCR), and their expression correlates with development of the symptoms of severe malaria. Structural studies revealed that PfEMP1 molecules present a helix-kinked-helix motif that forms the core of the EPCR-binding site. Using Rosetta-based design, we successfully grafted this motif onto a three-helical bundle scaffold. We show that this synthetic binder interacts with EPCR with nanomolar affinity and adopts the expected structure. We also assessed its ability to bind to antibodies found in immunized animals and in humans from malaria-endemic regions. Finally, we tested the capacity of the synthetic binder to effectively elicit antibodies that prevent EPCR binding and analyzed the degree of cross-reactivity of these antibodies across a diverse repertoire of EPCR-binding PfEMP1 proteins. Despite our synthetic binder adopting the correct structure, we find that it is not as effective as the CIDRα domain on which it is based for inducing adhesion-inhibitory antibodies. This cautions against the rational design of focused immunogens that contain the core features of a ligand-binding site of a protein family, rather than those of a neutralizing antibody epitope.IMPORTANCE Vaccines train our immune systems to generate antibodies which recognize pathogens. Some of these antibodies are highly protective, preventing infection, while others are ineffective. Structure-guided rational approaches allow design of synthetic molecules which contain only the regions of a pathogen required to induce production of protective antibodies. On the surfaces of red blood cells infected by the malaria parasite Plasmodium falciparum are parasite molecules called PfEMP1 proteins. PfEMP1 proteins, which bind to human receptor EPCR, are linked to development of severe malaria. We have designed a synthetic protein on which we grafted the EPCR-binding surface of a PfEMP1 protein. We use this molecule to show which fraction of protective antibodies recognize the EPCR-binding surface and test its effectiveness as a vaccine immunogen.
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Anticuerpos Antiprotozoarios/metabolismo , Receptor de Proteína C Endotelial/metabolismo , Proteínas/síntesis química , Proteínas/metabolismo , Proteínas Protozoarias/agonistas , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Secuencias de Aminoácidos , Animales , Anticuerpos Antiprotozoarios/inmunología , Sitios de Unión , Adhesión Celular , Receptor de Proteína C Endotelial/inmunología , Eritrocitos/parasitología , Humanos , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/prevención & control , Plasmodium falciparum/inmunología , Plasmodium falciparum/patogenicidad , Unión Proteica , Proteínas/química , Proteínas/inmunología , RatasRESUMEN
The deadly symptoms of malaria occur as Plasmodium parasites replicate within blood cells. Members of several variant surface protein families are expressed on infected blood cell surfaces. Of these, the largest and most ubiquitous are the Plasmodium-interspersed repeat (PIR) proteins, with more than 1,000 variants in some genomes. Their functions are mysterious, but differential pir gene expression associates with acute or chronic infection in a mouse malaria model. The membership of the PIR superfamily, and whether the family includes Plasmodium falciparum variant surface proteins, such as RIFINs and STEVORs, is controversial. Here we reveal the structure of the extracellular domain of a PIR from Plasmodium chabaudi We use structure-guided sequence analysis and molecular modeling to show that this fold is found across PIR proteins from mouse- and human-infective malaria parasites. Moreover, we show that RIFINs and STEVORs are not PIRs. This study provides a structure-guided definition of the PIRs and a molecular framework to understand their evolution.