RESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antiprotozoarios/inmunología , Eritrocitos/parasitología , Vacunas contra la Malaria/inmunología , Malaria Falciparum/inmunología , Plasmodium falciparum/inmunología , Adolescente , Adulto , Animales , Sitios de Unión , Proteínas Portadoras/inmunología , Reacciones Cruzadas/inmunología , Epítopos/inmunología , Femenino , Células HEK293 , Voluntarios Sanos , Humanos , Malaria Falciparum/parasitología , Masculino , Merozoítos/fisiología , Persona de Mediana Edad , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/inmunología , Conejos , Ratas , Ratas Sprague-Dawley , Adulto JovenRESUMEN
Candida albicans is a polymorphic fungus responsible for mucosal and skin infections. Candida cells establish themselves into biofilm communities resistant to most currently available antifungal agents. An increase of severe infections ensuing in fungal septic shock in elderly or immunosuppressed patients, along with the emergence of drug-resistant strains, urge the need for the development of alternative antifungal agents. In the search for novel antifungal drugs our laboratory demonstrated that two human ribonucleases from the vertebrate-specific RNaseA superfamily, hRNase3 and hRNase7, display a high anticandidal activity. In a previous work, we proved that the N-terminal region of the RNases was sufficient to reproduce most of the parental protein bactericidal activity. Next, we explored their potency against a fungal pathogen. Here, we have tested the N-terminal derived peptides that correspond to the eight human canonical RNases (RN1-8) against planktonic cells and biofilms of C. albicans. RN3 and RN7 peptides displayed the most potent inhibitory effect with a mechanism of action characterized by cell-wall binding, membrane permeabilization and biofilm eradication activities. Both peptides are able to eradicate planktonic and sessile cells, and to alter their gene expression, reinforcing its role as a lead candidate to develop novel antifungal and antibiofilm therapies.
Asunto(s)
Antifúngicos/farmacología , Candida albicans/efectos de los fármacos , Péptidos/química , Péptidos/farmacología , Ribonucleasas/química , Antifúngicos/química , Biopelículas/efectos de los fármacos , Candida albicans/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Pared Celular/efectos de los fármacos , Pared Celular/metabolismo , Proteína Catiónica del Eosinófilo/química , Proteína Catiónica del Eosinófilo/metabolismo , Proteína Catiónica del Eosinófilo/farmacología , Humanos , Péptidos/metabolismo , Ribonucleasas/metabolismo , Ribonucleasas/farmacologíaRESUMEN
Human RNase 6 is a cationic secreted protein that belongs to the RNase A superfamily. Its expression is induced in neutrophils and monocytes upon bacterial infection, suggesting a role in host defence. We present here the crystal structure of RNase 6 obtained at 1.72 Å (1 Å=0.1 nm) resolution, which is the first report for the protein 3D structure and thereby setting the basis for functional studies. The structure shows an overall kidney-shaped globular fold shared with the other known family members. Three sulfate anions bound to RNase 6 were found, interacting with residues at the main active site (His(15), His(122) and Gln(14)) and cationic surface-exposed residues (His(36), His(39), Arg(66) and His(67)). Kinetic characterization, together with prediction of protein-nucleotide complexes by molecular dynamics, was applied to analyse the RNase 6 substrate nitrogenous base and phosphate selectivity. Our results reveal that, although RNase 6 is a moderate catalyst in comparison with the pancreatic RNase type, its structure includes lineage-specific features that facilitate its activity towards polymeric nucleotide substrates. In particular, enzyme interactions at the substrate 5' end can provide an endonuclease-type cleavage pattern. Interestingly, the RNase 6 crystal structure revealed a novel secondary active site conformed by the His(36)-His(39) dyad that facilitates the polynucleotide substrate catalysis.
Asunto(s)
Ribonucleasa Pancreática/química , Ribonucleasa Pancreática/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Catálisis , Cristalografía por Rayos X , Humanos , Cinética , Datos de Secuencia Molecular , Unión Proteica , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
Eradication of established biofilm communities of pathogenic Gram-negative species is one of the pending challenges for the development of new antimicrobial agents. In particular, Pseudomonas aeruginosa is one of the main dreaded nosocomial species, with a tendency to form organized microbial communities that offer an enhanced resistance to conventional antibiotics. We describe here an engineered antimicrobial peptide (AMP) which combines bactericidal activity with a high bacterial cell agglutination and lipopolysaccharide (LPS) affinity. The RN3(5-17P22-36) peptide is a 30-mer derived from the eosinophil cationic protein (ECP), a host defense RNase secreted by eosinophils upon infection, with a wide spectrum of antipathogen activity. The protein displays high biofilm eradication activity that is not dependent on its RNase catalytic activity, as evaluated by using an active site-defective mutant. On the other hand, the peptide encompasses both the LPS-binding and aggregation-prone regions from the parental protein, which provide the appropriate structural features for the peptide's attachment to the bacterial exopolysaccharide layer and further improved removal of established biofilms. Moreover, the peptide's high cationicity and amphipathicity promote the cell membrane destabilization action. The results are also compared side by side with other reported AMPs effective against either planktonic and/or biofilm forms of Pseudomonas aeruginosa strain PAO1. The ECP and its derived peptide are unique in combining high bactericidal potency and cell agglutination activity, achieving effective biofilm eradication at a low micromolar range. We conclude that the designed RN3(5-17P22-36) peptide is a promising lead candidate against Gram-negative biofilms.
Asunto(s)
Antibacterianos/farmacología , Proteína Catiónica del Eosinófilo/química , Lipopolisacáridos/metabolismo , Pseudomonas aeruginosa/efectos de los fármacos , Aglutinación/efectos de los fármacos , Animales , Antibacterianos/metabolismo , Biopelículas/efectos de los fármacos , Eritrocitos/efectos de los fármacos , Humanos , Pruebas de Sensibilidad Microbiana , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Plancton/microbiología , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/fisiologíaRESUMEN
Human Ribonuclease 6 is a secreted protein belonging to the ribonuclease A (RNaseA) superfamily, a vertebrate specific family suggested to arise with an ancestral host defense role. Tissue distribution analysis revealed its expression in innate cell types, showing abundance in monocytes and neutrophils. Recent evidence of induction of the protein expression by bacterial infection suggested an antipathogen function in vivo. In our laboratory, the antimicrobial properties of the protein have been evaluated against Gram-negative and Gram-positive species and its mechanism of action was characterized using a membrane model. Interestingly, our results indicate that RNase6, as previously reported for RNase3, is able to specifically agglutinate Gram-negative bacteria as a main trait of its antimicrobial activity. Moreover, a side by side comparative analysis with the RN6(1-45) derived peptide highlights that the antimicrobial activity is mostly retained at the protein N-terminus. Further work by site directed mutagenesis and structural analysis has identified two residues involved in the protein antimicrobial action (Trp1 and Ile13) that are essential for the cell agglutination properties. This is the first structure-functional characterization of RNase6 antimicrobial properties, supporting its contribution to the infection focus clearance.
Asunto(s)
Antibacterianos/metabolismo , Infecciones Bacterianas/metabolismo , Fenómenos Fisiológicos Bacterianos , Exonucleasas/metabolismo , Aglutinación , Secuencia de Aminoácidos , Antibacterianos/química , Permeabilidad de la Membrana Celular , Pared Celular/fisiología , Exonucleasas/química , Humanos , Modelos Moleculares , Conformación Proteica , Alineación de SecuenciaRESUMEN
Antimicrobial proteins and peptides (AMPs) are important effectors of the innate immune system that play a vital role in the prevention of infections. Recent advances have highlighted the similarity between AMPs and amyloid proteins. Using the Eosinophil Cationic Protein as a model, we have rationalized the structure-activity relationships between amyloid aggregation and antimicrobial activity. Our results show how protein aggregation can induce bacteria agglutination and cell death. Using confocal and total internal reflection fluorescence microscopy we have tracked the formation in situ of protein amyloid-like aggregates at the bacteria surface and on membrane models. In both cases, fibrillar aggregates able to bind to amyloid diagnostic dyes were detected. Additionally, a single point mutation (Ile13 to Ala) can suppress the protein amyloid behavior, abolishing the agglutinating activity and impairing the antimicrobial action. The mutant is also defective in triggering both leakage and lipid vesicle aggregation. We conclude that ECP aggregation at the bacterial surface is essential for its cytotoxicity. Hence, we propose here a new prospective biological function for amyloid-like aggregates with potential biological relevance.
Asunto(s)
Amiloide/química , Antibacterianos/química , Bacterias/química , Proteína Catiónica del Eosinófilo/química , Inmunidad Innata , Viabilidad Microbiana , Sustitución de Aminoácidos , Amiloide/genética , Amiloide/inmunología , Antibacterianos/inmunología , Bacterias/genética , Bacterias/inmunología , Infecciones Bacterianas , Proteína Catiónica del Eosinófilo/genética , Proteína Catiónica del Eosinófilo/inmunología , Humanos , Mutación MissenseRESUMEN
Vertebrate secreted RNases (ribonucleases) are small proteins that play important roles in RNA metabolism, angiogenesis or host defence. In the present study we describe the antimicrobial properties of the N-terminal domain of the hcRNases (human canonical RNases) and show that their antimicrobial activity is well conserved among their lineage. Furthermore, all domains display a similar antimicrobial mechanism, characterized by bacteria agglutination followed by membrane permeabilization. The results of the present study show that, for all antimicrobial hcRNases, (i) activity is retained at the N-terminus and (ii) the antimicrobial mechanism is conserved. Moreover, using computational analysis we show that antimicrobial propensity may be conserved at the N-terminus for all vertebrate RNases, thereby suggesting that a defence mechanism could be a primary function in vertebrate RNases and that the N-terminus was selected to ensure this property. In a broader context, from the overall comparison of the peptides' physicochemical and biological properties, general correlation rules could be drawn to assist in the structure-based development of antimicrobial agents.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Bacterias/efectos de los fármacos , Ribonucleasas/química , Aglutinación , Secuencia de Aminoácidos , Animales , Péptidos Catiónicos Antimicrobianos/síntesis química , Péptidos Catiónicos Antimicrobianos/química , Bacterias/inmunología , Secuencia Conservada , Evolución Molecular , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Gramnegativas/inmunología , Bacterias Grampositivas/efectos de los fármacos , Bacterias Grampositivas/inmunología , Hemólisis , Interacciones Huésped-Patógeno , Humanos , Inmunidad Innata , Liposomas/química , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Fosfatidilcolinas/química , Fosfatidilgliceroles/química , Filogenia , Ribonucleasas/inmunología , Ribonucleasas/fisiología , OvinosRESUMEN
Plasmodium falciparum reticulocyte-binding protein homolog 5 (RH5) is a leading blood-stage malaria vaccine antigen target, currently in a phase 2b clinical trial as a full-length soluble protein/adjuvant vaccine candidate called RH5.1/Matrix-M. We identify that disordered regions of the full-length RH5 molecule induce non-growth inhibitory antibodies in human vaccinees and that a re-engineered and stabilized immunogen (including just the alpha-helical core of RH5) induces a qualitatively superior growth inhibitory antibody response in rats vaccinated with this protein formulated in Matrix-M adjuvant. In parallel, bioconjugation of this immunogen, termed "RH5.2," to hepatitis B surface antigen virus-like particles (VLPs) using the "plug-and-display" SpyTag-SpyCatcher platform technology also enables superior quantitative antibody immunogenicity over soluble protein/adjuvant in vaccinated mice and rats. These studies identify a blood-stage malaria vaccine candidate that may improve upon the current leading soluble protein vaccine candidate RH5.1/Matrix-M. The RH5.2-VLP/Matrix-M vaccine candidate is now under evaluation in phase 1a/b clinical trials.
Asunto(s)
Anticuerpos Antiprotozoarios , Vacunas contra la Malaria , Plasmodium falciparum , Proteínas Protozoarias , Vacunas de Partículas Similares a Virus , Animales , Vacunas contra la Malaria/inmunología , Anticuerpos Antiprotozoarios/inmunología , Plasmodium falciparum/inmunología , Vacunas de Partículas Similares a Virus/inmunología , Humanos , Ratones , Proteínas Protozoarias/inmunología , Ratas , Malaria Falciparum/prevención & control , Malaria Falciparum/inmunología , Antígenos de Protozoos/inmunología , Femenino , Proteínas Portadoras/inmunología , Ratones Endogámicos BALB CRESUMEN
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
BACKGROUND: A blood-stage Plasmodium falciparum malaria vaccine would provide a second line of defence to complement partially effective or waning immunity conferred by the approved pre-erythrocytic vaccines. RH5.1 is a soluble protein vaccine candidate for blood-stage P falciparum, formulated with Matrix-M adjuvant to assess safety and immunogenicity in a malaria-endemic adult and paediatric population for the first time. METHODS: We did a non-randomised, phase 1b, single-centre, dose-escalation, age de-escalation, first-in-human trial of RH5.1/Matrix-M in Bagamoyo, Tanzania. We recruited healthy adults (aged 18-45 years) and children (aged 5-17 months) to receive the RH5.1/Matrix-M vaccine candidate in the following three-dose regimens: 10 µg RH5.1 at 0, 1, and 2 months (Adults 10M), and the higher dose of 50 µg RH5.1 at 0 and 1 month and 10 µg RH5.1 at 6 months (delayed-fractional third dose regimen; Adults DFx). Children received either 10 µg RH5.1 at 0, 1, and 2 months (Children 10M) or 10 µg RH5.1 at 0, 1, and 6 months (delayed third dose regimen; Children 10D), and were recruited in parallel, followed by children who received the dose-escalation regimen (Children DFx) and children with higher malaria pre-exposure who also received the dose-escalation regimen (High Children DFx). All RH5.1 doses were formulated with 50 µg Matrix-M adjuvant. Primary outcomes for vaccine safety were solicited and unsolicited adverse events after each vaccination, along with any serious adverse events during the study period. The secondary outcome measures for immunogenicity were the concentration and avidity of anti-RH5.1 serum IgG antibodies and their percentage growth inhibition activity (GIA) in vitro, as well as cellular immunogenicity to RH5.1. All participants receiving at least one dose of vaccine were included in the primary analyses. This trial is registered at ClinicalTrials.gov, NCT04318002, and is now complete. FINDINGS: Between Jan 25, 2021, and April 15, 2021, we recruited 12 adults (six [50%] in the Adults 10M group and six [50%] in the Adults DFx group) and 48 children (12 each in the Children 10M, Children 10D, Children DFx, and High Children DFx groups). 57 (95%) of 60 participants completed the vaccination series and 55 (92%) completed 22 months of follow-up following the third vaccination. Vaccinations were well-tolerated across both age groups. There were five serious adverse events involving four child participants during the trial, none of which were deemed related to vaccination. RH5-specific T cell and serum IgG antibody responses were induced by vaccination and purified total IgG showed in vitro GIA against P falciparum. We found similar functional quality (ie, GIA per µg RH5-specific IgG) across all age groups and dosing regimens at 14 days after the final vaccination; the concentration of RH5.1-specific polyclonal IgG required to give 50% GIA was 14·3 µg/mL (95% CI 13·4-15·2). 11 children were vaccinated with the delayed third dose regimen and showed the highest median anti-RH5 serum IgG concentration 14 days following the third vaccination (723 µg/mL [IQR 511-1000]), resulting in all 11 who received the full series showing greater than 60% GIA following dilution of total IgG to 2·5 mg/mL (median 88% [IQR 81-94]). INTERPRETATION: The RH5.1/Matrix-M vaccine candidate shows an acceptable safety and reactogenicity profile in both adults and 5-17-month-old children residing in a malaria-endemic area, with all children in the delayed third dose regimen reaching a level of GIA previously associated with protective outcome against blood-stage P falciparum challenge in non-human primates. These data support onward efficacy assessment of this vaccine candidate against clinical malaria in young African children. FUNDING: The European and Developing Countries Clinical Trials Partnership; the UK Medical Research Council; the UK Department for International Development; the National Institute for Health and Care Research Oxford Biomedical Research Centre; the Division of Intramural Research, National Institute of Allergy and Infectious Diseases; the US Agency for International Development; and the Wellcome Trust.
RESUMEN
There is an urgent need to develop new agents against mycobacterial infections, such as tuberculosis and other respiratory tract or skin affections. In this study, we have tested two human antimicrobial RNases against mycobacteria. RNase 3, also called the eosinophil cationic protein, and RNase 7 are two small cationic proteins secreted by innate cells during host defense. Both proteins are induced upon infection displaying a wide range of antipathogen activities. In particular, they are released by leukocytes and epithelial cells, contributing to tissue protection. Here, the two RNases have been proven effective against Mycobacterium vaccae at a low micromolar level. High bactericidal activity correlated with their bacterial membrane depolarization and permeabilization activities. Further analysis on both protein-derived peptides identified for RNase 3 an N-terminus fragment that is even more active than the parental protein. Also, a potent bacterial agglutinating activity was unique to RNase 3 and its derived peptide. The particular biophysical properties of the RNase 3 active peptide are envisaged as a suitable reference for the development of novel antimycobacterial drugs. The results support the contribution of secreted RNases to the host immune response against mycobacteria.
Asunto(s)
Antibacterianos/farmacología , Proteína Catiónica del Eosinófilo/farmacología , Mycobacterium/efectos de los fármacos , Ribonucleasas/farmacología , Secuencia de Aminoácidos , Antibacterianos/síntesis química , Membrana Celular/efectos de los fármacos , Permeabilidad de la Membrana Celular , Activación Enzimática , Proteína Catiónica del Eosinófilo/síntesis química , Genes Sintéticos , Humanos , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Datos de Secuencia Molecular , Proteínas Recombinantes/farmacología , Ribonucleasas/síntesis químicaRESUMEN
SUMMARY: AMPA is a web application for assessing the antimicrobial domains of proteins, with a focus on the design on new antimicrobial drugs. The application provides fast discovery of antimicrobial patterns in proteins that can be used to develop new peptide-based drugs against pathogens. Results are shown in a user-friendly graphical interface and can be downloaded as raw data for later examination. AVAILABILITY: AMPA is freely available on the web at http://tcoffee.crg.cat/apps/ampa. The source code is also available in the web. CONTACT: marc.torrent@upf.edu; david.andreu@upf.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Asunto(s)
Algoritmos , Antiinfecciosos/química , Péptidos/química , Péptidos Catiónicos Antimicrobianos/química , Internet , Lenguajes de Programación , Estructura Terciaria de Proteína , Programas InformáticosRESUMEN
The human eosinophil cationic protein (ECP), also known as RNase 3, is an eosinophil secretion protein that is involved in innate immunity and displays antipathogen and proinflammatory activities. ECP has a high binding affinity for heterosaccharides, such as bacterial lipopolysaccharides and heparan sulfate found in the glycocalix of eukaryotic cells. We have crystallized ECP in complex with sulfate anions in a new monoclinic crystal form. In this form, the active site groove is exposed, providing an alternative model for ligand binding studies. By exploring the protein-sulfate complex, we have defined the sulfate binding site architecture. Three main sites (S1-S3) are located in the protein active site; S1 and S2 overlap with the phosphate binding sites involved in RNase nucleotide recognition. A new site (S3) that is unique to ECP is one of the key anchoring points for sulfated ligands. Arg 1 and Arg 7 in S3, together with Arg 34 and Arg 36 in S1, form the main basic clusters that assist in the recognition of ligand anionic groups. The location of additional sulfate bound molecules, some of which contribute to the crystal packing, may mimic the binding to extended anionic polymers. In conclusion, the structural data define a binding pattern for the recognition of sulfated molecules that can modulate the role of ECP in innate immunity. The results reveal the structural basis for the high affinity of ECP for glycosaminoglycans and can assist in structure-based drug design of inhibitors of the protein cytotoxicity to host tissues during inflammation.
Asunto(s)
Proteína Catiónica del Eosinófilo/química , Sulfatos/química , Secuencia de Aminoácidos , Dominio Catalítico , Cristalización , Proteína Catiónica del Eosinófilo/metabolismo , Glicosaminoglicanos/química , Glicosaminoglicanos/metabolismo , Humanos , Ligandos , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Antimicrobial proteins and peptides (AMPs) are essential effectors of innate immunity, acting as a first line of defense against bacterial infections. Many AMPs exhibit high affinity for cell wall structures such as lipopolysaccharide (LPS), a potent endotoxin able to induce sepsis. Hence, understanding how AMPs can interact with and neutralize LPS endotoxin is of special relevance for human health. Eosinophil cationic protein (ECP) is an eosinophil secreted protein with high activity against both Gram-negative and Gram-positive bacteria. ECP has a remarkable affinity for LPS and a distinctive agglutinating activity. By using a battery of LPS-truncated E. coli mutant strains, we demonstrate that the polysaccharide moiety of LPS is essential for ECP-mediated bacterial agglutination, thereby modulating its antimicrobial action. The mechanism of action of ECP at the bacterial surface is drastically affected by the LPS structure and in particular by its polysaccharide moiety. We have also analyzed an N-terminal fragment that retains the whole protein activity and displays similar cell agglutination behavior. Conversely, a fragment with further minimization of the antimicrobial domain, though retaining the antimicrobial capacity, significantly loses its agglutinating activity, exhibiting a different mechanism of action which is not dependent on the LPS composition. The results highlight the correlation between the protein's antimicrobial activity and its ability to interact with the LPS outer layer and promote bacterial agglutination.
Asunto(s)
Pared Celular/química , Proteína Catiónica del Eosinófilo/farmacología , Escherichia coli/efectos de los fármacos , Lipopolisacáridos/química , Oligopéptidos/farmacología , Aglutinación/efectos de los fármacos , Adhesión Bacteriana/efectos de los fármacos , Sitios de Unión , Conformación de Carbohidratos , Proteína Catiónica del Eosinófilo/química , Proteína Catiónica del Eosinófilo/metabolismo , Eosinófilos/citología , Eosinófilos/inmunología , Eosinófilos/metabolismo , Escherichia coli/genética , Escherichia coli/fisiología , Humanos , Lipopolisacáridos/metabolismo , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Modelos Moleculares , Oligopéptidos/química , Oligopéptidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Relación Estructura-ActividadRESUMEN
Antimicrobial RNases are small cationic proteins belonging to the vertebrate RNase A superfamily and endowed with a wide range of antipathogen activities. Vertebrate RNases, while sharing the active site architecture, are found to display a variety of noncatalytical biological properties, providing an excellent example of multitask proteins. The antibacterial activity of distant related RNases suggested that the family evolved from an ancestral host-defence function. The review provides a structural insight into antimicrobial RNases, taking as a reference the human RNase 3, also named eosinophil cationic protein (ECP). A particular high binding affinity against bacterial wall structures mediates the protein action. In particular, the interaction with the lipopolysaccharides at the Gram-negative outer membrane correlates with the protein antimicrobial and specific cell agglutinating activity. Although a direct mechanical action at the bacteria wall seems to be sufficient to trigger bacterial death, a potential intracellular target cannot be discarded. Indeed, the cationic clusters at the protein surface may serve both to interact with nucleic acids and cell surface heterosaccharides. Sequence determinants for ECP activity were screened by prediction tools, proteolysis and peptide synthesis. Docking results are complementing the structural analysis to delineate the protein anchoring sites for anionic targets of biological significance.
Asunto(s)
Antibacterianos/química , Proteína Catiónica del Eosinófilo/química , Factores Inmunológicos/química , Secuencia de Aminoácidos , Animales , Antibacterianos/inmunología , Fenómenos Fisiológicos Bacterianos , Proteína Catiónica del Eosinófilo/inmunología , Interacciones Huésped-Patógeno , Humanos , Inmunidad Innata , Factores Inmunológicos/inmunología , Modelos Moleculares , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
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.
Asunto(s)
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
Background: RH5 is the leading vaccine candidate for the Plasmodium falciparum blood stage and has shown impact on parasite growth in the blood in a human clinical trial. RH5 binds to Ripr and CyRPA at the apical end of the invasive merozoite form, and this complex, designated RCR, is essential for entry into human erythrocytes. RH5 has advanced to human clinical trials, and the impact on parasite growth in the blood was encouraging but modest. This study assessed the potential of a protein-in-adjuvant blood stage malaria vaccine based on a combination of RH5, Ripr and CyRPA to provide improved neutralizing activity against P. falciparum in vitro. Methods: Mice were immunized with the individual RCR antigens to down select the best performing adjuvant formulation and rats were immunized with the individual RCR antigens to select the correct antigen dose. A second cohort of rats were immunized with single, double and triple antigen combinations to assess immunogenicity and parasite neutralizing activity in growth inhibition assays. Results: The DPX® platform was identified as the best performing formulation in potentiating P. falciparum inhibitory antibody responses to these antigens. The three antigens derived from RH5, Ripr and CyRPA proteins formulated with DPX induced highly inhibitory parasite neutralising antibodies. Notably, RH5 either as a single antigen or in combination with Ripr and/or CyRPA, induced inhibitory antibodies that outperformed CyRPA, Ripr. Conclusion: An RCR combination vaccine may not induce substantially improved protective immunity as compared with RH5 as a single immunogen in a clinical setting and leaves the development pathway open for other antigens to be combined with RH5 as a next generation malaria vaccine.
Asunto(s)
Vacunas contra la Malaria , Malaria Falciparum , Humanos , Ratones , Ratas , Animales , Antígenos de Protozoos , Proteínas Protozoarias/metabolismo , Malaria Falciparum/parasitología , Plasmodium falciparum , Anticuerpos Antiprotozoarios , Vacunas CombinadasRESUMEN
BACKGROUND: There is an ongoing global effort to design, manufacture, and clinically assess vaccines against SARS-CoV-2. Over the course of the ongoing pandemic a number of new SARS-CoV-2 virus isolates or variants of concern (VoC) have been identified containing mutations in key proteins. METHODS: In this study we describe the generation and preclinical assessment of a ChAdOx1-vectored vaccine (AZD2816) which expresses the spike protein of the Beta VoC (B.1.351). FINDINGS: We demonstrate that AZD2816 is immunogenic after a single dose. When AZD2816 is used as a booster dose in animals primed with a vaccine encoding the original spike protein (ChAdOx1 nCoV-19/ [AZD1222]), an increase in binding and neutralising antibodies against Beta (B.1.351), Gamma (P.1) and Delta (B.1.617.2) is observed following each additional dose. In addition, a strong and polyfunctional T cell response was measured all booster regimens. INTERPRETATION: Real world data is demonstrating that one or more doses of licensed SARS-CoV-2 vaccines confer reduced protection against hospitalisation and deaths caused by divergent VoC, including Omicron. Our data support the ongoing clinical development and testing of booster vaccines to increase immunity against highly mutated VoC. FUNDING: This research was funded by AstraZeneca with supporting funds from MRC and BBSRC.
Asunto(s)
COVID-19 , SARS-CoV-2 , COVID-19/prevención & control , Vacunas contra la COVID-19 , ChAdOx1 nCoV-19 , Humanos , SARS-CoV-2/genéticaRESUMEN
BackgroundAlthough recent epidemiological data suggest that pneumococci may contribute to the risk of SARS-CoV-2 disease, cases of coinfection with Streptococcus pneumoniae in patients with coronavirus disease 2019 (COVID-19) during hospitalization have been reported infrequently. This apparent contradiction may be explained by interactions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and pneumococci in the upper airway, resulting in the escape of SARS-CoV-2 from protective host immune responses.MethodsHere, we investigated the relationship of these 2 respiratory pathogens in 2 distinct cohorts of health care workers with asymptomatic or mildly symptomatic SARS-CoV-2 infection identified by systematic screening and patients with moderate to severe disease who presented to the hospital. We assessed the effect of coinfection on host antibody, cellular, and inflammatory responses to the virus.ResultsIn both cohorts, pneumococcal colonization was associated with diminished antiviral immune responses, which primarily affected mucosal IgA levels among individuals with mild or asymptomatic infection and cellular memory responses in infected patients.ConclusionOur findings suggest that S. pneumoniae impair host immunity to SARS-CoV-2 and raise the question of whether pneumococcal carriage also enables immune escape of other respiratory viruses and facilitates reinfection.Trial registrationISRCTN89159899 (FASTER study) and ClinicalTrials.gov NCT03502291 (LAIV study).
Asunto(s)
COVID-19 , SARS-CoV-2 , Personal de Salud , Humanos , Inmunidad , Streptococcus pneumoniaeRESUMEN
Basigin, or CD147, has been reported as a coreceptor used by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to invade host cells. Basigin also has a well-established role in Plasmodium falciparum malaria infection of human erythrocytes, where it is bound by one of the parasite's invasion ligands, reticulocyte binding protein homolog 5 (RH5). Here, we sought to validate the claim that the receptor binding domain (RBD) of SARS-CoV-2 spike glycoprotein can form a complex with basigin, using RH5-basigin as a positive control. Using recombinantly expressed proteins, size exclusion chromatography and surface plasmon resonance, we show that neither RBD nor full-length spike glycoprotein bind to recombinant human basigin (expressed in either Escherichia coli or mammalian cells). Further, polyclonal anti-basigin IgG did not block SARS-CoV-2 infection of Vero E6 cells. Given the immense interest in SARS-CoV-2 therapeutic targets to improve treatment options for those who become seriously ill with coronavirus disease 2019 (COVID-19), we would caution the inclusion of basigin in this list on the basis of its reported direct interaction with SARS-CoV-2 spike glycoprotein. IMPORTANCE Reducing the mortality and morbidity associated with COVID-19 remains a global health priority. Vaccines have proven highly effective at preventing infection and hospitalization, but efforts must continue to improve treatment options for those who still become seriously ill. Critical to these efforts is the identification of host factors that are essential to viral entry and replication. Basigin, or CD147, was previously identified as a possible therapeutic target based on the observation that it may act as a coreceptor for SARS-CoV-2, binding to the receptor binding domain of the spike protein. Here, we show that there is no direct interaction between the RBD and basigin, casting doubt on its role as a coreceptor and plausibility as a therapeutic target.