RESUMO
This work continues a series of recently published studies that employ recombinant single-domain antibody (sdAb, or nanobody®) generation technologies to battle viruses by a passive immunization approach. As a proof of principle, we describe a modified technique to efficiently generate protective molecules against a particular strain of influenza virus within a reasonably short period of time. This approach starts with the immunization of a camel (Camelus bactrianus) with the specified antigen-enriched material presented in as natural a form as possible. An avian influenza virus A/Mallard/Pennsylvania/10218/84 (H5N2) adapted for mice was used as a model source of antigens for both the immunization and phage display-based selection procedures. To significantly increase activities of initially selected monovalent single-domain antibodies, we propose a new type of sdAb formatting that involves the addition of a special type of coiled-coil sequence, the isoleucine zipper domain (ILZ). Presumably, the ILZ-containing peptides adopt trimeric parallel conformations. After the formatting, the biological activities (virus neutralization) of the initially selected anti-influenza virus (H5N2) sdAbs were significantly increased. Intraperitoneal or intranasal administration of the formatted sdAb at 2h before or 24h after viral challenge specifically protects mice from lethal infection with influenza virus. We hope that the described approach combined with the selection focused on particular conservative epitopes will lead to the generation of sdAb-based molecules protective against a broad spectrum of influenza virus subtypes.
Assuntos
Anticorpos Antivirais/imunologia , Técnicas Imunológicas/métodos , Vírus da Influenza A Subtipo H5N2/fisiologia , Influenza Humana/prevenção & controle , Anticorpos de Domínio Único/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/administração & dosagem , Anticorpos Antivirais/química , Anticorpos Antivirais/genética , Camelus/genética , Camelus/imunologia , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Vírus da Influenza A Subtipo H5N2/efeitos dos fármacos , Vírus da Influenza A Subtipo H5N2/genética , Influenza Humana/imunologia , Influenza Humana/virologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Anticorpos de Domínio Único/administração & dosagem , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/genéticaRESUMO
One effective method for the prevention and treatment of influenza infection is passive immunization. In our study, we examined the feasibility of creating an antibody-based preparation with a prolonged protective effect against influenza virus. Single-domain antibodies (sdAbs) specific for influenza virus hemagglutinin were generated. Experiments in mouse models showed 100% survivability for both intranasal sdAbs administration 24h prior to influenza challenge and 24h after infection. sdAb-gene delivery by an adenoviral vector led to gene expression for up to 14days. Protection by a recombinant adenovirus containing the sdAb gene was observed in cases of administration prior to influenza infection (14d-24h). We also demonstrated that the single administration of a combined preparation containing sdAb DNA and protein expanded the protection time window from 14d prior to 48h after influenza infection. This approach and the application of a broad-spectrum sdAbs will allow the development of efficient drugs for the prevention and treatment of viral infections produced by pandemic virus variants and other infections.
Assuntos
Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A Subtipo H5N2/imunologia , Influenza Humana/prevenção & controle , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/imunologia , Adenoviridae/genética , Adenoviridae/imunologia , Animais , Linhagem Celular , Feminino , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Humanos , Imunização Passiva , Vírus da Influenza A Subtipo H5N2/genética , Influenza Humana/imunologia , Influenza Humana/virologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
The studies of ELCCT influence on spontaneous and KCl-stimulated 3H-dopamine release by synaptosomes isolated from the rabbit brain hemispheres and diencephalic-brainstem areas are presented in this paper. Kinetic characteristics of the releasing system (Bm and Vo) were shown to decrease at earlier stages (15 minutes to 2 hrs) and increase at later stages (1 to 14 days). No substantial difference was shown in release responses of the synaptosomes derived from brain hemispheres and diencephalic-brainstem regions. The data suggest the major alterations in the mechanisms of dopamine release in ELCCT.
