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.
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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
In quantum mechanical many-body systems, long-range and anisotropic interactions promote rich spatial structure and can lead to quantum frustration, giving rise to a wealth of complex, strongly correlated quantum phases1. Long-range interactions play an important role in nature; however, quantum simulations of lattice systems have largely not been able to realize such interactions. A wide range of efforts are underway to explore long-range interacting lattice systems using polar molecules2-5, Rydberg atoms2,6-8, optical cavities9-11 or magnetic atoms12-15. Here we realize novel quantum phases in a strongly correlated lattice system with long-range dipolar interactions using ultracold magnetic erbium atoms. As we tune the dipolar interaction to be the dominant energy scale in our system, we observe quantum phase transitions from a superfluid into dipolar quantum solids, which we directly detect using quantum gas microscopy with accordion lattices. Controlling the interaction anisotropy by orienting the dipoles enables us to realize a variety of stripe-ordered states. Furthermore, by transitioning non-adiabatically through the strongly correlated regime, we observe the emergence of a range of metastable stripe-ordered states. This work demonstrates that novel strongly correlated quantum phases can be realized using long-range dipolar interactions in optical lattices, opening the door to quantum simulations of a wide range of lattice models with long-range and anisotropic interactions.
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The Coalition for Epidemic Preparedness Innovations' "100-day moonshot" aspires to launch a new vaccine within 100 days of pathogen identification, followed by large-scale vaccine availability within the "second hundred days." Here, we describe work to optimize adenoviral vector manufacturing for rapid response, by minimizing time to clinical trial and first large-scale supply, and maximizing output from the available manufacturing footprint. We describe a rapid virus seed expansion workflow that allows vaccine release to clinical trials within 60 days of antigen sequence identification, followed by vaccine release from globally distributed sites within a further 40 days. We also describe a perfusion-based upstream production process, designed to maximize output while retaining simplicity and suitability for existing manufacturing facilities. This improves upstream volumetric productivity of ChAdOx1 nCoV-19 by approximately fourfold and remains compatible with the existing downstream process, yielding drug substance sufficient for 10,000 doses from each liter of bioreactor capacity. This accelerated manufacturing process, along with other advantages such as thermal stability, supports the ongoing value of adenovirus-vectored vaccines as a rapidly adaptable and deployable platform for emergency response.
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Adenoviridae , Vacunas contra el Adenovirus , Humanos , Adenoviridae/genética , ChAdOx1 nCoV-19 , Reactores Biológicos , Brotes de Enfermedades/prevención & controlRESUMEN
BACKGROUND: Sporozoite invasion of hepatocytes is an essential step in the Plasmodium life-cycle and has similarities, at the cellular level, to merozoite invasion of erythrocytes. In the case of the Plasmodium blood-stage, efforts to identify host-pathogen protein-protein interactions have yielded important insights including vaccine candidates. In the case of sporozoite-hepatocyte invasion, the host-pathogen protein-protein interactions involved are poorly understood. METHODS: To gain a better understanding of the protein-protein interaction between the sporozoite ligands and host receptors, a systematic screen was performed. The previous Plasmodium falciparum and human surface protein ectodomain libraries were substantially extended, resulting in the creation of new libraries comprising 88 P. falciparum sporozoite protein coding sequences and 182 sequences encoding human hepatocyte surface proteins. Having expressed recombinant proteins from these sequences, a plate-based assay was used, capable of detecting low affinity interactions between recombinant proteins, modified for enhanced throughput, to screen the proteins for interactions. The novel interactions identified in the screen were characterized biochemically, and their essential role in parasite invasion was further elucidated using antibodies and genetically manipulated Plasmodium parasites. RESULTS: A total of 7540 sporozoite-hepatocyte protein pairs were tested under conditions capable of detecting interactions of at least 1.2 µM KD. An interaction between the human fibroblast growth factor receptor 4 (FGFR4) and the P. falciparum protein Pf34 is identified and reported here, characterizing its affinity and demonstrating the blockade of the interaction by reagents, including a monoclonal antibody. Furthermore, further interactions between Pf34 and a second P. falciparum rhoptry neck protein, PfRON6, and between human low-density lipoprotein receptor (LDLR) and the P. falciparum protein PIESP15 are identified. Conditional genetic deletion confirmed the essentiality of PfRON6 in the blood-stage, consistent with the important role of this protein in parasite lifecycle. Pf34 was refractory to attempted genetic modification. Antibodies to Pf34 abrogated the interaction and had a modest effect upon sporozoite invasion into primary human hepatocytes. CONCLUSION: Pf34 and PfRON6 may be members of a functionally important invasion complex which could be a target for future interventions. The modified interaction screening assay, protein expression libraries and P. falciparum mutant parasites reported here may be a useful tool for protein interaction discovery and antigen candidate screening which could be of wider value to the scientific community.
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Hepatocitos , Plasmodium falciparum , Proteínas Protozoarias , Esporozoítos , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Hepatocitos/parasitología , Humanos , Esporozoítos/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Interacciones Huésped-Patógeno , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Interacciones Huésped-Parásitos , Unión ProteicaRESUMEN
BACKGROUND: A new variant of SARS-CoV-2, B.1.1.7, emerged as the dominant cause of COVID-19 disease in the UK from November, 2020. We report a post-hoc analysis of the efficacy of the adenoviral vector vaccine, ChAdOx1 nCoV-19 (AZD1222), against this variant. METHODS: Volunteers (aged ≥18 years) who were enrolled in phase 2/3 vaccine efficacy studies in the UK, and who were randomly assigned (1:1) to receive ChAdOx1 nCoV-19 or a meningococcal conjugate control (MenACWY) vaccine, provided upper airway swabs on a weekly basis and also if they developed symptoms of COVID-19 disease (a cough, a fever of 37·8°C or higher, shortness of breath, anosmia, or ageusia). Swabs were tested by nucleic acid amplification test (NAAT) for SARS-CoV-2 and positive samples were sequenced through the COVID-19 Genomics UK consortium. Neutralising antibody responses were measured using a live-virus microneutralisation assay against the B.1.1.7 lineage and a canonical non-B.1.1.7 lineage (Victoria). The efficacy analysis included symptomatic COVID-19 in seronegative participants with a NAAT positive swab more than 14 days after a second dose of vaccine. Participants were analysed according to vaccine received. Vaccine efficacy was calculated as 1 - relative risk (ChAdOx1 nCoV-19 vs MenACWY groups) derived from a robust Poisson regression model. This study is continuing and is registered with ClinicalTrials.gov, NCT04400838, and ISRCTN, 15281137. FINDINGS: Participants in efficacy cohorts were recruited between May 31 and Nov 13, 2020, and received booster doses between Aug 3 and Dec 30, 2020. Of 8534 participants in the primary efficacy cohort, 6636 (78%) were aged 18-55 years and 5065 (59%) were female. Between Oct 1, 2020, and Jan 14, 2021, 520 participants developed SARS-CoV-2 infection. 1466 NAAT positive nose and throat swabs were collected from these participants during the trial. Of these, 401 swabs from 311 participants were successfully sequenced. Laboratory virus neutralisation activity by vaccine-induced antibodies was lower against the B.1.1.7 variant than against the Victoria lineage (geometric mean ratio 8·9, 95% CI 7·2-11·0). Clinical vaccine efficacy against symptomatic NAAT positive infection was 70·4% (95% CI 43·6-84·5) for B.1.1.7 and 81·5% (67·9-89·4) for non-B.1.1.7 lineages. INTERPRETATION: ChAdOx1 nCoV-19 showed reduced neutralisation activity against the B.1.1.7 variant compared with a non-B.1.1.7 variant in vitro, but the vaccine showed efficacy against the B.1.1.7 variant of SARS-CoV-2. FUNDING: UK Research and Innovation, National Institute for Health Research (NIHR), Coalition for Epidemic Preparedness Innovations, NIHR Oxford Biomedical Research Centre, Thames Valley and South Midlands NIHR Clinical Research Network, and AstraZeneca.
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Anticuerpos Neutralizantes/sangre , Vacunas contra la COVID-19/inmunología , COVID-19/prevención & control , COVID-19/virología , SARS-CoV-2/inmunología , Adolescente , Adulto , COVID-19/epidemiología , Prueba de Ácido Nucleico para COVID-19 , Vacunas contra la COVID-19/efectos adversos , ChAdOx1 nCoV-19 , Femenino , Humanos , Masculino , Persona de Mediana Edad , Técnicas de Amplificación de Ácido Nucleico , Pandemias/prevención & control , Método Simple Ciego , Reino Unido/epidemiología , Carga Viral , Adulto JovenRESUMEN
Manufacturing has been the key factor limiting rollout of vaccination during the COVID-19 pandemic, requiring rapid development and large-scale implementation of novel manufacturing technologies. ChAdOx1 nCoV-19 (AZD1222, Vaxzevria) is an efficacious vaccine against SARS-CoV-2, based upon an adenovirus vector. We describe the development of a process for the production of this vaccine and others based upon the same platform, including novel features to facilitate very large-scale production. We discuss the process economics and the "distributed manufacturing" approach we have taken to provide the vaccine at globally-relevant scale and with international security of supply. Together, these approaches have enabled the largest viral vector manufacturing campaign to date, providing a substantial proportion of global COVID-19 vaccine supply at low cost.
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Vacunas contra la COVID-19 , COVID-19/prevención & control , ChAdOx1 nCoV-19 , Industria Farmacéutica/métodos , Desarrollo de Vacunas , Animales , Escherichia coli , Geografía , Células HEK293 , Humanos , Pan troglodytes , SARS-CoV-2 , Tecnología Farmacéutica , Vacunación/instrumentaciónRESUMEN
Sickle cell disease significantly impacts one's quality of life (QOL); thus, randomized controlled trials (RCTs) have integrated patient-reported outcomes (PROs) to assess patients' health from their perspective. We aim to evaluate the completeness of reporting of PROs included in sickle cell disease RCTs. We searched MEDLINE, Embase and Cochrane Central Register of Controlled Trials (CENTRAL) for published sickle cell disease RCTs with at least one PRO measure from 2006 to 2021. In a masked, duplicate fashion, two investigators evaluated RCTs using the Consolidated Standards of Reporting in Trials (CONSORT)-PRO adaptation and Cochrane Collaboration Risk of Bias (RoB) 2.0 tool. The primary objective was mean percent completeness of the CONSORT-PRO adaptation. Additional relationships between trial characteristics and completeness of reporting were evaluated. Mean completeness of reporting of RCTs was 41.49% (SD = 20.90). Randomized controlled trials with primary outcomes were more complete (57.50%, SD = 8.33) than RCTs with secondary PROs (33.48%, SD = 20.91). We did not find a significant difference in completion between trials with primary PROs and secondary PROs (t1 = 2.07; p = 0.06). Our secondary objectives included factors that may be associated with completeness of PRO reporting. Of the 12 included studies, five were considered to be overall 'high' RoB (41.67%). In each of the five domains, the majority of studies received 'low' RoB evaluations. Incomplete PRO reporting was common within sickle cell RCTs. Therefore, we recommend future RCTs including PROs should take measures to increase completeness of reporting.
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Medición de Resultados Informados por el Paciente , Calidad de Vida , Humanos , Ensayos Clínicos Controlados Aleatorios como AsuntoRESUMEN
BACKGROUND: The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) might be curtailed by vaccination. We assessed the safety, reactogenicity, and immunogenicity of a viral vectored coronavirus vaccine that expresses the spike protein of SARS-CoV-2. METHODS: We did a phase 1/2, single-blind, randomised controlled trial in five trial sites in the UK of a chimpanzee adenovirus-vectored vaccine (ChAdOx1 nCoV-19) expressing the SARS-CoV-2 spike protein compared with a meningococcal conjugate vaccine (MenACWY) as control. Healthy adults aged 18-55 years with no history of laboratory confirmed SARS-CoV-2 infection or of COVID-19-like symptoms were randomly assigned (1:1) to receive ChAdOx1 nCoV-19 at a dose of 5â×â1010 viral particles or MenACWY as a single intramuscular injection. A protocol amendment in two of the five sites allowed prophylactic paracetamol to be administered before vaccination. Ten participants assigned to a non-randomised, unblinded ChAdOx1 nCoV-19 prime-boost group received a two-dose schedule, with the booster vaccine administered 28 days after the first dose. Humoral responses at baseline and following vaccination were assessed using a standardised total IgG ELISA against trimeric SARS-CoV-2 spike protein, a muliplexed immunoassay, three live SARS-CoV-2 neutralisation assays (a 50% plaque reduction neutralisation assay [PRNT50]; a microneutralisation assay [MNA50, MNA80, and MNA90]; and Marburg VN), and a pseudovirus neutralisation assay. Cellular responses were assessed using an ex-vivo interferon-γ enzyme-linked immunospot assay. The co-primary outcomes are to assess efficacy, as measured by cases of symptomatic virologically confirmed COVID-19, and safety, as measured by the occurrence of serious adverse events. Analyses were done by group allocation in participants who received the vaccine. Safety was assessed over 28 days after vaccination. Here, we report the preliminary findings on safety, reactogenicity, and cellular and humoral immune responses. The study is ongoing, and was registered at ISRCTN, 15281137, and ClinicalTrials.gov, NCT04324606. FINDINGS: Between April 23 and May 21, 2020, 1077 participants were enrolled and assigned to receive either ChAdOx1 nCoV-19 (n=543) or MenACWY (n=534), ten of whom were enrolled in the non-randomised ChAdOx1 nCoV-19 prime-boost group. Local and systemic reactions were more common in the ChAdOx1 nCoV-19 group and many were reduced by use of prophylactic paracetamol, including pain, feeling feverish, chills, muscle ache, headache, and malaise (all p<0·05). There were no serious adverse events related to ChAdOx1 nCoV-19. In the ChAdOx1 nCoV-19 group, spike-specific T-cell responses peaked on day 14 (median 856 spot-forming cells per million peripheral blood mononuclear cells, IQR 493-1802; n=43). Anti-spike IgG responses rose by day 28 (median 157 ELISA units [EU], 96-317; n=127), and were boosted following a second dose (639 EU, 360-792; n=10). Neutralising antibody responses against SARS-CoV-2 were detected in 32 (91%) of 35 participants after a single dose when measured in MNA80 and in 35 (100%) participants when measured in PRNT50. After a booster dose, all participants had neutralising activity (nine of nine in MNA80 at day 42 and ten of ten in Marburg VN on day 56). Neutralising antibody responses correlated strongly with antibody levels measured by ELISA (R2=0·67 by Marburg VN; p<0·001). INTERPRETATION: ChAdOx1 nCoV-19 showed an acceptable safety profile, and homologous boosting increased antibody responses. These results, together with the induction of both humoral and cellular immune responses, support large-scale evaluation of this candidate vaccine in an ongoing phase 3 programme. FUNDING: UK Research and Innovation, Coalition for Epidemic Preparedness Innovations, National Institute for Health Research (NIHR), NIHR Oxford Biomedical Research Centre, Thames Valley and South Midland's NIHR Clinical Research Network, and the German Center for Infection Research (DZIF), Partner site Gießen-Marburg-Langen.
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Betacoronavirus/inmunología , Infecciones por Coronavirus/prevención & control , Inmunogenicidad Vacunal , Pandemias/prevención & control , Neumonía Viral/prevención & control , Vacunas Virales/efectos adversos , Vacunas Virales/inmunología , Acetaminofén/uso terapéutico , Adenovirus de los Simios/genética , Adulto , Analgésicos no Narcóticos/uso terapéutico , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , COVID-19 , Vacunas contra la COVID-19 , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/inmunología , Femenino , Vectores Genéticos/administración & dosificación , Humanos , Inmunización Secundaria , Inmunoglobulina G/sangre , Masculino , Neumonía Viral/tratamiento farmacológico , SARS-CoV-2 , Método Simple Ciego , Glicoproteína de la Espiga del Coronavirus/inmunología , Linfocitos T/inmunología , Reino Unido , Vacunas Virales/administración & dosificaciónRESUMEN
While a number of amphibian skin microbiomes have been characterized, it is unclear how these communities might vary in response to seasonal changes in the environment and the corresponding behaviors that many amphibians exhibit. Given recent studies demonstrating the importance of the skin microbiome in frog innate immune defense against pathogens, investigating how changes in the environment impact the microbial species present will provide a better understanding of conditions that may alter host susceptibility to pathogens in their environment. We sampled the bacterial skin microbiome of North American wood frogs (Rana sylvatica) from two breeding ponds in the spring, along with the bacterial community present in their vernal breeding pools, and frogs from the nearby forest floor in the summer and fall to determine whether community composition differs by sex, vernal pond site, or temporally across season (spring, summer, fall). Taxon relative abundance data reveals a profile of bacterial phyla similar to those previously described on anuran skin, with Proteobacteria, Bacteroidetes, and Actinobacteria dominating the wood frog skin microbiome. Our results indicate that sex had no significant effect on skin microbiota diversity; however, this may be due to our limited female frog sample size. Vernal pool site had a small but significant effect on skin microbiota, but skin-associated communities were more similar to each other than to the communities observed in the frogs' respective pond water. Across seasons, diversity analyses suggest that there are significant differences between the bacterial skin microbiome of frogs from spring and summer/fall groups while the average α-diversity per frog remained consistent. These results illustrate seasonal variation in wood frog skin microbiome structure and highlight the importance of considering temporal trends in an amphibian microbiome, particularly for species whose life history requires recurrent shifts in habitat and behavior.
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Actinobacteria/aislamiento & purificación , Bacteroidetes/aislamiento & purificación , Proteobacteria/aislamiento & purificación , Ranidae/microbiología , Piel/microbiología , Actinobacteria/clasificación , Actinobacteria/genética , Animales , Bacteroidetes/clasificación , Bacteroidetes/genética , ADN Bacteriano/genética , Microbiota/genética , Estanques , Proteobacteria/clasificación , Proteobacteria/genética , ARN Ribosómico 16S/genética , Estaciones del Año , Estados UnidosRESUMEN
BACKGROUND: Cardiac assessment in noncardiac surgery clinical practice guidelines should be supported by the highest-quality evidence such as that offered by systematic reviews. Currently, the methodological and reporting quality of these studies remains unknown. METHODS: We used PubMed to search for all clinical practice guidelines related to perioperative cardiovascular patients undergoing noncardiac surgery from 2010 to 2021. The included clinical practice guidelines were analysed for all systematic reviews and meta-analyses. The primary objective of this study was to determine reporting and methodological quality using the PRISMA (Preferred Reporting Instrument for Systematic Reviews and Meta-Analyses) and AMSTAR-2 (A Measurement Tool to Assess Systematic Reviews-2) instruments. Our secondary objective was to compare systematic reviews conducted by the Cochrane Collaboration with non-Cochrane studies. RESULTS: Three clinical practice guidelines were included in our study. Within these, 78 systematic reviews were included. PRISMA completion ranged from 34.8% to 100.0% with a mean of 76.9%. AMSTAR-2 completion ranged from 15.6% to 96.9% with a mean of 58.0%. Fifty-four systematic reviews underpinned a clinical practice guidelines recommendation, of which 25 were rated 'critically low' by AMSTAR-2 appraisal. Cochrane systematic reviews typically performed better than non-Cochrane studies, but were a minority of the included studies (10/78). CONCLUSION: We found deficiencies in several key areas regarding the methodological and reporting qualities of systematic reviews included in cardiac assessment in noncardiac surgery clinical practice guidelines. As these clinical practice guidelines are instrumental to clinical decision-making and patient care in cardiac assessment in noncardiac surgery, we advocate for improved reporting quality among systematic reviews cited as supportive evidence for these recommendations.
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Enfermedades Cardiovasculares/diagnóstico , Enfermedades Cardiovasculares/prevención & control , Guías de Práctica Clínica como Asunto , Proyectos de Investigación/normas , Procedimientos Quirúrgicos Operativos , Revisiones Sistemáticas como Asunto/normas , Humanos , Metaanálisis como Asunto , Medición de Riesgo , Revisiones Sistemáticas como Asunto/métodosRESUMEN
Invasion of host erythrocytes is essential to the life cycle of Plasmodium parasites and development of the pathology of malaria. The stages of erythrocyte invasion, including initial contact, apical reorientation, junction formation, and active invagination, are directed by coordinated release of specialized apical organelles and their parasite protein contents. Among these proteins, and central to invasion by all species, are two parasite protein families, the reticulocyte-binding protein homologue (RH) and erythrocyte-binding like proteins, which mediate host-parasite interactions. RH5 from Plasmodium falciparum (PfRH5) is the only member of either family demonstrated to be necessary for erythrocyte invasion in all tested strains, through its interaction with the erythrocyte surface protein basigin (also known as CD147 and EMMPRIN). Antibodies targeting PfRH5 or basigin efficiently block parasite invasion in vitro, making PfRH5 an excellent vaccine candidate. Here we present crystal structures of PfRH5 in complex with basigin and two distinct inhibitory antibodies. PfRH5 adopts a novel fold in which two three-helical bundles come together in a kite-like architecture, presenting binding sites for basigin and inhibitory antibodies at one tip. This provides the first structural insight into erythrocyte binding by the Plasmodium RH protein family and identifies novel inhibitory epitopes to guide design of a new generation of vaccines against the blood-stage parasite.
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Anticuerpos Bloqueadores/química , Basigina/química , Eritrocitos/química , Malaria , Plasmodium falciparum/química , Anticuerpos Bloqueadores/inmunología , Antígenos de Protozoos/química , Antígenos de Protozoos/inmunología , Basigina/inmunología , Sitios de Unión , Cristalografía por Rayos X , Epítopos/química , Epítopos/inmunología , Interacciones Huésped-Parásitos/inmunología , Humanos , Malaria/parasitología , Modelos Moleculares , Plasmodium falciparum/inmunología , Proteínas Protozoarias/química , Proteínas Protozoarias/inmunologíaRESUMEN
There is great interest in the development of Ab-inducing subunit vaccines targeting infections, including HIV, malaria, and Ebola. We previously reported that adenovirus vectored vaccines are potent in priming Ab responses, but uncertainty remains regarding the optimal approach for induction of humoral immune responses. In this study, using OVA as a model Ag, we assessed the magnitude of the primary and anamnestic Ag-specific IgG responses of mice to four clinically relevant vaccine formulations: replication-deficient adenovirus; modified vaccinia Ankara (a poxvirus); protein with alum; and protein in the squalene oil-in-water adjuvant Addavax. We then used flow cytometric assays capable of measuring total and Ag-specific germinal center (GC) B cell and follicular Th cell responses to compare the induction of these responses by the different formulations. We report that adenovirus vectored vaccines induce Ag insert-specific GC B cell and Ab responses of a magnitude comparable to those induced by a potent protein/squalene oil-in-water formulation whereas-despite a robust overall GC response-the insert-specific GC B cell and Ab responses induced by modified vaccinia Ankara were extremely weak. Ag-specific follicular Th cell responses to adenovirus vectored vaccines exceeded those induced by other platforms at day 7 after immunization. We found little evidence that innate immune activation by adenovirus may act as an adjuvant in such a manner that the humoral response to a recombinant protein may be enhanced by coadministering with an adenovirus lacking a transgene of interest. Overall, these studies provide further support for the use of replication-deficient adenoviruses to induce humoral responses.
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Adenoviridae/inmunología , Adyuvantes Inmunológicos/farmacología , Linfocitos B/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Vacunas Virales/inmunología , Animales , Antígenos/inmunología , Western Blotting , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Vectores Genéticos , Centro Germinal/inmunología , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ovalbúmina/inmunologíaRESUMEN
BACKGROUND: Models of controlled human malaria infection (CHMI) initiated by mosquito bite have been widely used to assess efficacy of preerythrocytic vaccine candidates in small proof-of-concept phase 2a clinical trials. Efficacy testing of blood-stage malaria parasite vaccines, however, has generally relied on larger-scale phase 2b field trials in malaria-endemic populations. We report the use of a blood-stage P. falciparum CHMI model to assess blood-stage vaccine candidates, using their impact on the parasite multiplication rate (PMR) as the primary efficacy end point. METHODS: Fifteen healthy United Kingdom adult volunteers were vaccinated with FMP2.1, a protein vaccine that is based on the 3D7 clone sequence of apical membrane antigen 1 (AMA1) and formulated in Adjuvant System 01 (AS01). Twelve vaccinees and 15 infectivity controls subsequently underwent blood-stage CHMI. Parasitemia was monitored by quantitative real-time polymerase chain reaction (PCR) analysis, and PMR was modeled from these data. RESULTS: FMP2.1/AS01 elicited anti-AMA1 T-cell and serum antibody responses. Analysis of purified immunoglobulin G showed functional growth inhibitory activity against P. falciparum in vitro. There were no vaccine- or CHMI-related safety concerns. All volunteers developed blood-stage parasitemia, with no impact of the vaccine on PMR. CONCLUSIONS: FMP2.1/AS01 demonstrated no efficacy after blood-stage CHMI. However, the model induced highly reproducible infection in all volunteers and will accelerate proof-of-concept testing of future blood-stage vaccine candidates. CLINICAL TRIALS REGISTRATION: NCT02044198.
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Antígenos de Protozoos/inmunología , Vacunas contra la Malaria/inmunología , Malaria Falciparum/prevención & control , Proteínas de la Membrana/inmunología , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Adulto , Ensayo de Immunospot Ligado a Enzimas , Eritrocitos/parasitología , Femenino , Humanos , Inmunogenicidad Vacunal , Estadios del Ciclo de Vida , Malaria Falciparum/parasitología , Masculino , Persona de Mediana Edad , Modelos Biológicos , Plasmodium falciparum/fisiología , Adulto JovenRESUMEN
There is intense interest in induction and characterization of strain-transcending neutralizing Ab against antigenically variable human pathogens. We have recently identified the human malaria parasite Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) as a target of broadly neutralizing Abs, but there is little information regarding the functional mechanism(s) of Ab-mediated neutralization. In this study, we report that vaccine-induced polyclonal anti-PfRH5 Abs inhibit the tight attachment of merozoites to erythrocytes and are capable of blocking the interaction of PfRH5 with its receptor basigin. Furthermore, by developing anti-PfRH5 mAbs, we provide evidence of the following: 1) the ability to block the PfRH5-basigin interaction in vitro is predictive of functional activity, but absence of blockade does not predict absence of functional activity; 2) neutralizing mAbs bind spatially related epitopes on the folded protein, involving at least two defined regions of the PfRH5 primary sequence; 3) a brief exposure window of PfRH5 is likely to necessitate rapid binding of Ab to neutralize parasites; and 4) intact bivalent IgG contributes to but is not necessary for parasite neutralization. These data provide important insight into the mechanisms of broadly neutralizing anti-malaria Abs and further encourage anti-PfRH5-based malaria prevention efforts.
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Anticuerpos Neutralizantes/inmunología , Anticuerpos Antiprotozoarios/inmunología , Proteínas Portadoras/inmunología , Merozoítos/inmunología , Plasmodium falciparum/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Antiprotozoarios/metabolismo , Proteínas Portadoras/metabolismo , Mapeo Epitopo , Epítopos/química , Epítopos/inmunología , Eritrocitos/inmunología , Eritrocitos/parasitología , Humanos , Cinética , Vacunas contra la Malaria/inmunología , Malaria Falciparum/inmunología , Ratones , Pruebas de Neutralización , Plasmodium falciparum/crecimiento & desarrollo , Unión Proteica/inmunología , ConejosRESUMEN
BACKGROUND: Circumsporozoite protein (CS) is the antigenic target for RTS,S, the most advanced malaria vaccine to date. Heterologous prime-boost with the viral vectors simian adenovirus 63 (ChAd63)-modified vaccinia virus Ankara (MVA) is the most potent inducer of T-cells in humans, demonstrating significant efficacy when expressing the preerythrocytic antigen insert multiple epitope-thrombospondin-related adhesion protein (ME-TRAP). We hypothesized that ChAd63-MVA containing CS may result in a significant clinical protective efficacy. METHODS: We conducted an open-label, 2-site, partially randomized Plasmodium falciparum sporozoite controlled human malaria infection (CHMI) study to compare the clinical efficacy of ChAd63-MVA CS with ChAd63-MVA ME-TRAP. RESULTS: One of 15 vaccinees (7%) receiving ChAd63-MVA CS and 2 of 15 (13%) receiving ChAd63-MVA ME-TRAP achieved sterile protection after CHMI. Three of 15 vaccinees (20%) receiving ChAd63-MVA CS and 5 of 15 (33%) receiving ChAd63-MVA ME-TRAP demonstrated a delay in time to treatment, compared with unvaccinated controls. In quantitative polymerase chain reaction analyses, ChAd63-MVA CS was estimated to reduce the liver parasite burden by 69%-79%, compared with 79%-84% for ChAd63-MVA ME-TRAP. CONCLUSIONS: ChAd63-MVA CS does reduce the liver parasite burden, but ChAd63-MVA ME-TRAP remains the most promising antigenic insert for a vectored liver-stage vaccine. Detailed analyses of parasite kinetics may allow detection of smaller but biologically important differences in vaccine efficacy that can influence future vaccine development. CLINICAL TRIALS REGISTRATION: NCT01623557.
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Vacunas contra la Malaria/inmunología , Malaria Falciparum/prevención & control , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Adenovirus de los Simios/genética , Adenovirus de los Simios/inmunología , Adolescente , Adulto , Anticuerpos Antiprotozoarios/biosíntesis , Epítopos/inmunología , Femenino , Vectores Genéticos , Humanos , Interferón gamma/inmunología , Hígado/virología , Malaria Falciparum/inmunología , Malaria Falciparum/parasitología , Masculino , Persona de Mediana Edad , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , Adulto JovenRESUMEN
BACKGROUND: Controlled human malaria infection (CHMI) studies increasingly rely on nucleic acid test (NAT) methods to detect and quantify parasites in the blood of infected participants. The lower limits of detection and quantification vary amongst the assays used throughout the world, which may affect the ability of mathematical models to accurately estimate the liver-to-blood inoculum (LBI) values that are used to judge the efficacy of pre-erythrocytic vaccine and drug candidates. METHODS: Samples were collected around the time of onset of pre-patent parasitaemia from subjects who enrolled in two different CHMI clinical trials. Blood samples were tested for Plasmodium falciparum 18S rRNA and/or rDNA targets by different NAT methods and results were compared. Methods included an ultrasensitive, large volume modification of an established quantitative reverse transcription PCR (qRT-PCR) assay that achieves detection of as little as one parasite/mL of whole blood. RESULTS: Large volume qRT-PCR at the University of Washington was the most sensitive test and generated quantifiable data more often than any other NAT methodology. Standard quantitative PCR (qPCR) performed at the University of Oxford and standard volume qRT-PCR performed at the University of Washington were less sensitive than the large volume qRT-PCR, especially at 6.5 days after CHMI. In these trials, the proportion of participants for whom LBI could be accurately quantified using parasite density value greater than or equal to the lower limit of quantification was increased. A greater improvement would be expected in trials in which numerous subjects receive a lower LBI or low dose challenge. CONCLUSIONS: Standard qPCR and qRT-PCR methods with analytical sensitivities of ~20 parasites/mL probably suffice for most CHMI purposes, but the newly developed large volume qRT-PCR may be able to answer specific questions when more analytical sensitivity is required.
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Malaria/diagnóstico , Malaria/parasitología , Reacción en Cadena de la Polimerasa/métodos , Adulto , ADN Protozoario/sangre , Femenino , Humanos , Límite de Detección , Malaria/epidemiología , Masculino , Persona de Mediana Edad , Técnicas de Diagnóstico Molecular , Parasitemia/diagnóstico , Parasitemia/parasitología , ARN Ribosómico 18S/genética , Adulto JovenRESUMEN
The development of effective vaccines against difficult disease targets will require the identification of new subunit vaccination strategies that can induce and maintain effective immune responses in humans. Here we report on a phase 1a clinical trial using the AMA1 antigen from the blood-stage Plasmodium falciparum malaria parasite delivered either as recombinant protein formulated with Alhydrogel adjuvant with and without CPG 7909, or using recombinant vectored vaccines--chimpanzee adenovirus ChAd63 and the orthopoxvirus MVA. A variety of promising "mixed-modality" regimens were tested. All volunteers were primed with ChAd63, and then subsequently boosted with MVA and/or protein-in-adjuvant using either an 8- or 16-week prime-boost interval. We report on the safety of these regimens, as well as the T cell, B cell, and serum antibody responses. Notably, IgG antibody responses primed by ChAd63 were comparably boosted by AMA1 protein vaccine, irrespective of whether CPG 7909 was included in the Alhydrogel adjuvant. The ability to improve the potency of a relatively weak aluminium-based adjuvant in humans, by previously priming with an adenoviral vaccine vector encoding the same antigen, thus offers a novel vaccination strategy for difficult or neglected disease targets when access to more potent adjuvants is not possible.
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Adyuvantes Inmunológicos/administración & dosificación , Antígenos de Protozoos/administración & dosificación , Vacunas contra la Malaria/administración & dosificación , Malaria Falciparum/prevención & control , Plasmodium falciparum/inmunología , Adenovirus de los Simios/genética , Adulto , Hidróxido de Aluminio/administración & dosificación , Antígenos de Protozoos/inmunología , Terapia Combinada , Vectores Genéticos/administración & dosificación , Humanos , Inmunización Secundaria , Masculino , Persona de Mediana Edad , Oligodesoxirribonucleótidos/administración & dosificación , Orthopoxvirus/genética , Vacunación , Adulto JovenRESUMEN
No vaccine has yet proven effective against the blood-stages of Plasmodium falciparum, which cause the symptoms and severe manifestations of malaria. We recently found that PfRH5, a P. falciparum-specific protein expressed in merozoites, is efficiently targeted by broadly-neutralizing, vaccine-induced antibodies. Here we show that antibodies against PfRH5 efficiently inhibit the in vitro growth of short-term-adapted parasite isolates from Cambodia, and that the EC(50) values of antigen-specific antibodies against PfRH5 are lower than those against PfAMA1. Since antibody responses elicited by multiple antigens are speculated to improve the efficacy of blood-stage vaccines, we conducted detailed assessments of parasite growth inhibition by antibodies against PfRH5 in combination with antibodies against seven other merozoite antigens. We found that antibodies against PfRH5 act synergistically with antibodies against certain other merozoite antigens, most notably with antibodies against other erythrocyte-binding antigens such as PfRH4, to inhibit the growth of a homologous P. falciparum clone. A combination of antibodies against PfRH4 and basigin, the erythrocyte receptor for PfRH5, also potently inhibited parasite growth. This methodology provides the first quantitative evidence that polyclonal vaccine-induced antibodies can act synergistically against P. falciparum antigens and should help to guide the rational development of future multi-antigen vaccines.
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Anticuerpos Monoclonales de Origen Murino/inmunología , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/inmunología , Proteínas Portadoras/inmunología , Eritrocitos/inmunología , Merozoítos/inmunología , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Animales , Eritrocitos/parasitología , Humanos , Vacunas contra la Malaria/inmunología , Malaria Falciparum/inmunología , Malaria Falciparum/prevención & control , RatonesRESUMEN
Controlled human malaria infection is used to measure efficacy of candidate malaria vaccines before field studies are undertaken. Mathematical modeling using data from quantitative polymerase chain reaction (qPCR) parasitemia monitoring can discriminate between vaccine effects on the parasite's liver and blood stages. Uncertainty regarding the most appropriate modeling method hinders interpretation of such trials. We used qPCR data from 267 Plasmodium falciparum infections to compare linear, sine-wave, and normal-cumulative-density-function models. We find that the parameters estimated by these models are closely correlated, and their predictive accuracy for omitted data points was similar. We propose that future studies include the linear model.
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Hígado/parasitología , Vacunas contra la Malaria/farmacología , Malaria Falciparum/parasitología , Modelos Biológicos , Parasitemia/parasitología , Plasmodium falciparum/efectos de los fármacos , Animales , Humanos , Hígado/efectos de los fármacos , Hígado/inmunología , Vacunas contra la Malaria/sangre , Vacunas contra la Malaria/inmunología , Malaria Falciparum/genética , Malaria Falciparum/inmunología , Malaria Falciparum/prevención & control , Parasitemia/genética , Parasitemia/inmunología , Parasitemia/prevención & control , Plasmodium falciparum/genética , Plasmodium falciparum/inmunologíaRESUMEN
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.