Assuntos
Toxinas Botulínicas/genética , Toxinas Botulínicas/uso terapêutico , Botulismo/terapia , Paralisia/terapia , Animais , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Toxinas Botulínicas/imunologia , Camelídeos Americanos/imunologia , Modelos Animais de Doenças , Sistemas de Liberação de Medicamentos , Cobaias , Humanos , Macaca , Camundongos , Engenharia de ProteínasRESUMO
Highly pathogenic avian H5 influenza viruses persist among poultry and wild birds throughout the world. They sometimes cause interspecies transmission between avian and mammalian hosts. H5 viruses possessing the HA of subclade 2.3.4.4, 2.3.2.1, 2.2.1, or 7.2 were detected between 2015 and 2018. To understand the neutralizing epitopes of H5-HA, we characterized 15 human monoclonal antibodies (mAbs) against the HA of H5 viruses, which were obtained from volunteers who received the H5N1 vaccine that contains a subclade 2.2.1 or 2.1.3.2 virus as an antigen. Twelve mAbs were specific for the HA of subclade 2.2.1, two mAbs were specific for the HA of subclade 2.1.3.2, and one mAb was specific for the HA of both. Of the 15 mAbs analyzed, nine, which were specific for the HA of subclade 2.2.1, and shared the VH and VL genes, possessed hemagglutination inhibition and neutralizing activities, whereas the others did not. A single amino acid substitution or insertion at positions 144-147 in antigenic site A conferred resistance against these nine mAbs to the subclade 2.2.1 viruses. The amino acids at positions 144-147 are highly conserved among subclade 2.2.1, but differ from those of other subclades. These results show that the neutralizing epitope including amino acids at positions 144-147 is targeted by human antibodies, and plays a role in the antigenic difference between subclade 2.2.1 and other subclades.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A/imunologia , Aminoácidos , Anticorpos Monoclonais/genética , Anticorpos Neutralizantes/genética , Anticorpos Antivirais/genética , Mapeamento de Epitopos , Epitopos , Testes de Inibição da Hemaglutinação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Evasão da Resposta Imune/genética , Virus da Influenza A Subtipo H5N1/imunologia , Vírus da Influenza A/genética , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/imunologia , Mutação , Testes de NeutralizaçãoRESUMO
Previously unidentified viruses, such as Middle East respiratory syndrome coronavirus, continue to emerge and threaten populations, while powerful new techniques have identified many new human and animal viruses. Similarly, existing viruses, from Ebola virus to chikungunya virus, are reemerging and spreading to new geographical regions. These viruses often pose a challenge for researchers to study due to their highly pathogenic nature. Lentiviral and rhabdoviral pseudotypes are excellent tools for studying enveloped viruses and have contributed to many recent advances in areas such as receptor usage, viral entry and serology. In particular, pseudotypes allow the safe study of unknown or highly pathogenic viruses. They also allow the initial characterization of aspects of infection such as cellular tropism for difficult to culture viruses. In this review we will introduce various pseudotyping systems for emerging viruses, including chikungunya virus, Ebola virus, SARS and MERS coronaviruses and Nipah virus, as well as their use in diverse studies including drug screening and antibody neutralization. We will also discuss the limitations and potential caveats using pseudotypes.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Terapia Genética/métodos , Vetores Genéticos , Proteínas do Envelope Viral/genética , Tropismo Viral/fisiologia , Anticorpos Neutralizantes/genética , Coronavirus/genética , Ebolavirus/genética , Vírus Nipah/genética , Vacinas ViraisRESUMO
Monoclonal antibodies (mAbs) that bind and neutralize human pathogens have great therapeutic potential. Advances in automated screening and liquid handling have resulted in the ability to discover antigen-specific antibodies either directly from human blood or from various combinatorial libraries (phage, bacteria, or yeast). There remain, however, bottlenecks in the cloning, expression and evaluation of such lead antibodies identified in primary screens that hinder high-throughput screening. As such, "hit-to-lead identification" remains both expensive and time-consuming. By combining the advantages of overlap extension PCR (OE-PCR) and a genetically stable yet easily manipulatable microbial expression host Pichia pastoris, we have developed an automated pipeline for the rapid production and screening of full-length antigen-specific mAbs. Here, we demonstrate the speed, feasibility and cost-effectiveness of our approach by generating several broadly neutralizing antibodies against human immunodeficiency virus (HIV).
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , HIV/imunologia , Pichia/metabolismo , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Neutralizantes/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Anticorpos Anti-HIV/genética , Anticorpos Anti-HIV/isolamento & purificação , Anticorpos Anti-HIV/metabolismo , Humanos , Pichia/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Fatores de TempoRESUMO
Influenza causes serious and sometimes fatal disease in individuals at risk due to advanced age or immunodeficiencies. Despite progress in the development of seasonal influenza vaccines, vaccine efficacy in elderly and immunocompromised individuals remains low. We recently developed a passive immunization strategy using an adeno-associated virus (AAV) vector to deliver a neutralizing anti-influenza antibody at the site of infection, the nasal airways. Here we show that young, old, and immunodeficient (severe combined immunodeficient [SCID]) mice that were treated intranasally with AAV9 vector expressing a modified version of the broadly neutralizing anti-influenza antibody FI6 were protected and exhibited no signs of disease following an intranasal challenge with the mouse-adapted H1N1 influenza strain A/Puerto Rico/8/1934(H1N1) (PR8) (Mt. Sinai strain). Nonvaccinated mice succumbed to the PR8 challenge due to severe weight loss. We propose that airway-directed AAV9 passive immunization against airborne infectious agents may be beneficial in elderly and immunocompromised patients, for whom there still exists an unmet need for effective vaccination against influenza.