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1.
Cell Rep ; 42(11): 113330, 2023 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-38007690

RESUMO

IGHV3-33-encoded antibodies are prevalent in the human humoral response against the Plasmodium falciparum circumsporozoite protein (PfCSP). Among VH3-33 antibodies, cross-reactivity between PfCSP major repeat (NANP), minor (NVDP), and junctional (NPDP) motifs is associated with high affinity and potent parasite inhibition. However, the molecular basis of antibody cross-reactivity and the relationship with efficacy remain unresolved. Here, we perform an extensive structure-function characterization of 12 VH3-33 anti-PfCSP monoclonal antibodies (mAbs) with varying degrees of cross-reactivity induced by immunization of mice expressing a human immunoglobulin gene repertoire. We identify residues in the antibody paratope that mediate cross-reactive binding and delineate four distinct epitope conformations induced by antibody binding, with one consistently associated with high protective efficacy and another that confers comparably potent inhibition of parasite liver invasion. Our data show a link between molecular features of cross-reactive VH3-33 mAb binding to PfCSP and mAb potency, relevant for the development of antibody-based interventions against malaria.


Assuntos
Malária Falciparum , Malária , Camundongos , Humanos , Animais , Plasmodium falciparum/genética , Anticorpos Antiprotozoários , Proteínas de Protozoários/genética , Epitopos , Anticorpos Monoclonais , Malária Falciparum/parasitologia
3.
NPJ Vaccines ; 8(1): 52, 2023 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-37029167

RESUMO

The development of an effective and durable vaccine remains a central goal in the fight against malaria. Circumsporozoite protein (CSP) is the major surface protein of sporozoites and the target of the only licensed Plasmodium falciparum (Pf) malaria vaccine, RTS,S/AS01. However, vaccine efficacy is low and short-lived, highlighting the need for a second-generation vaccine with superior efficacy and durability. Here, we report a Helicobacter pylori apoferritin-based nanoparticle immunogen that elicits strong B cell responses against PfCSP epitopes that are targeted by the most potent human monoclonal antibodies. Glycan engineering of the scaffold and fusion of an exogenous T cell epitope enhanced the anti-PfCSP B cell response eliciting strong, long-lived and protective humoral immunity in mice. Our study highlights the power of rational vaccine design to generate a highly efficacious second-generation anti-infective malaria vaccine candidate and provides the basis for its further development.

4.
Elife ; 122023 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-36971345

RESUMO

Immunoglobulin loci-transgenic animals are widely used in antibody discovery and increasingly in vaccine response modelling. In this study, we phenotypically characterised B-cell populations from the Intelliselect Transgenic mouse (Kymouse) demonstrating full B-cell development competence. Comparison of the naïve B-cell receptor (BCR) repertoires of Kymice BCRs, naïve human, and murine BCR repertoires revealed key differences in germline gene usage and junctional diversification. These differences result in Kymice having CDRH3 length and diversity intermediate between mice and humans. To compare the structural space explored by CDRH3s in each species' repertoire, we used computational structure prediction to show that Kymouse naïve BCR repertoires are more human-like than mouse-like in their predicted distribution of CDRH3 shape. Our combined sequence and structural analysis indicates that the naïve Kymouse BCR repertoire is diverse with key similarities to human repertoires, while immunophenotyping confirms that selected naïve B cells are able to go through complete development.


Assuntos
Anticorpos , Linfócitos B , Animais , Humanos , Camundongos , Camundongos Transgênicos , Imunofenotipagem , Sequenciamento de Nucleotídeos em Larga Escala , Receptores de Antígenos de Linfócitos B/genética
5.
PLoS Pathog ; 18(11): e1010999, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36441829

RESUMO

Antibodies targeting the human malaria parasite Plasmodium falciparum circumsporozoite protein (PfCSP) can prevent infection and disease. PfCSP contains multiple central repeating NANP motifs; some of the most potent anti-infective antibodies against malaria bind to these repeats. Multiple antibodies can bind the repeating epitopes concurrently by engaging into homotypic Fab-Fab interactions, which results in the ordering of the otherwise largely disordered central repeat into a spiral. Here, we characterize IGHV3-33/IGKV1-5-encoded monoclonal antibody (mAb) 850 elicited by immunization of transgenic mice with human immunoglobulin loci. mAb 850 binds repeating NANP motifs with picomolar affinity, potently inhibits Plasmodium falciparum (Pf) in vitro and, when passively administered in a mouse challenge model, reduces liver burden to a similar extent as some of the most potent anti-PfCSP mAbs yet described. Like other IGHV3-33/IGKV1-5-encoded anti-NANP antibodies, mAb 850 primarily utilizes its HCDR3 and germline-encoded aromatic residues to recognize its core NANP motif. Biophysical and cryo-electron microscopy analyses reveal that up to 19 copies of Fab 850 can bind the PfCSP repeat simultaneously, and extensive homotypic interactions are observed between densely-packed PfCSP-bound Fabs to indirectly improve affinity to the antigen. Together, our study expands on the molecular understanding of repeat-induced homotypic interactions in the B cell response against PfCSP for potently protective mAbs against Pf infection.


Assuntos
Vacinas Antimaláricas , Malária Falciparum , Malária , Humanos , Camundongos , Animais , Plasmodium falciparum , Microscopia Crioeletrônica , Malária Falciparum/parasitologia , Proteínas de Protozoários , Malária/parasitologia , Camundongos Transgênicos , Anticorpos Monoclonais , Anticorpos Antiprotozoários
6.
PLoS Pathog ; 16(3): e1008373, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32150583

RESUMO

Lasting protection has long been a goal for malaria vaccines. The major surface antigen on Plasmodium falciparum sporozoites, the circumsporozoite protein (PfCSP), has been an attractive target for vaccine development and most protective antibodies studied to date interact with the central NANP repeat region of PfCSP. However, it remains unclear what structural and functional characteristics correlate with better protection by one antibody over another. Binding to the junctional region between the N-terminal domain and central NANP repeats has been proposed to result in superior protection: this region initiates with the only NPDP sequence followed immediately by NANP. Here, we isolated antibodies in Kymab mice immunized with full-length recombinant PfCSP and two protective antibodies were selected for further study with reactivity against the junctional region. X-ray and EM structures of two monoclonal antibodies, mAb667 and mAb668, shed light on their differential affinity and specificity for the junctional region. Importantly, these antibodies also bind to the NANP repeat region with equal or better affinity. A comparison with an NANP-only binding antibody (mAb317) revealed roughly similar but statistically distinct levels of protection against sporozoite challenge in mouse liver burden models, suggesting that junctional antibody protection might relate to the ability to also cross-react with the NANP repeat region. Our findings indicate that additional efforts are necessary to isolate a true junctional antibody with no or much reduced affinity to the NANP region to elucidate the role of the junctional epitope in protection.


Assuntos
Anticorpos Monoclonais Murinos/química , Anticorpos Antiprotozoários/química , Sítios de Ligação de Anticorpos , Epitopos/química , Plasmodium falciparum/química , Proteínas de Protozoários/química , Animais , Anticorpos Monoclonais Murinos/imunologia , Anticorpos Antiprotozoários/imunologia , Epitopos/imunologia , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Relação Estrutura-Atividade
7.
Influenza Other Respir Viruses ; 13(6): 556-563, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31536169

RESUMO

BACKGROUND: The extent of transmission of influenza in hospital settings is poorly understood. Next generation sequencing may improve this by providing information on the genetic relatedness of viral strains. OBJECTIVES: We aimed to apply next generation sequencing to describe transmission in hospital and compare with methods based on routinely-collected data. METHODS: All influenza samples taken through routine care from patients at University College London Hospitals NHS Foundation Trust (September 2012 to March 2014) were included. We conducted Illumina sequencing and identified genetic clusters. We compared nosocomial transmission estimates defined using classical methods (based on time from admission to sample) and genetic clustering. We identified pairs of cases with space-time links and assessed genetic relatedness. RESULTS: We sequenced influenza sampled from 214 patients. There were 180 unique genetic strains, 16 (8.8%) of which seeded a new transmission chain. Nosocomial transmission was indicated for 32 (15.0%) cases using the classical definition and 34 (15.8%) based on genetic clustering. Of the 50 patients in a genetic cluster, 11 (22.0%) had known space-time links with other cases in the same cluster. Genetic distances between pairs of cases with space-time links were lower than for pairs without spatial links (P < .001). CONCLUSIONS: Genetic data confirmed that nosocomial transmission contributes significantly to the hospital burden of influenza and elucidated transmission chains. Prospective next generation sequencing could support outbreak investigations and monitor the impact of infection and control measures.


Assuntos
Infecção Hospitalar/transmissão , Infecção Hospitalar/virologia , Influenza Humana/transmissão , Influenza Humana/virologia , Orthomyxoviridae/fisiologia , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Análise por Conglomerados , Infecção Hospitalar/epidemiologia , Estudos Transversais , Feminino , Genoma Viral/genética , Hospitais , Humanos , Controle de Infecções , Influenza Humana/epidemiologia , Londres/epidemiologia , Masculino , Pessoa de Meia-Idade , Orthomyxoviridae/classificação , Orthomyxoviridae/genética , RNA Viral/genética , Estudos Retrospectivos , Análise de Sequência de DNA , Adulto Jovem
8.
Virus Res ; 239: 10-16, 2017 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-27497916

RESUMO

The BEEHIVE (Bridging the Evolution and Epidemiology of HIV in Europe) project aims to analyse nearly-complete viral genomes from >3000 HIV-1 infected Europeans using high-throughput deep sequencing techniques to investigate the virus genetic contribution to virulence. Following the development of a computational pipeline, including a new de novo assembler for RNA virus genomes, to generate larger contiguous sequences (contigs) from the abundance of short sequence reads that characterise the data, another area that determines genome sequencing success is the quality and quantity of the input RNA. A pilot experiment with 125 patient plasma samples was performed to investigate the optimal method for isolation of HIV-1 viral RNA for long amplicon genome sequencing. Manual isolation with the QIAamp Viral RNA Mini Kit (Qiagen) was superior over robotically extracted RNA using either the QIAcube robotic system, the mSample Preparation Systems RNA kit with automated extraction by the m2000sp system (Abbott Molecular), or the MagNA Pure 96 System in combination with the MagNA Pure 96 Instrument (Roche Diagnostics). We scored amplification of a set of four HIV-1 amplicons of ∼1.9, 3.6, 3.0 and 3.5kb, and subsequent recovery of near-complete viral genomes. Subsequently, 616 BEEHIVE patient samples were analysed to determine factors that influence successful amplification of the genome in four overlapping amplicons using the QIAamp Viral RNA Kit for viral RNA isolation. Both low plasma viral load and high sample age (stored before 1999) negatively influenced the amplification of viral amplicons >3kb. A plasma viral load of >100,000 copies/ml resulted in successful amplification of all four amplicons for 86% of the samples, this value dropped to only 46% for samples with viral loads of <20,000 copies/ml.


Assuntos
Genoma Viral , Genômica , Infecções por HIV/virologia , HIV-1/genética , RNA Viral , Genômica/métodos , Genótipo , HIV-1/classificação , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , RNA Viral/isolamento & purificação , Carga Viral , Sequenciamento Completo do Genoma
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