RESUMO
In addition to vaccines, noninfectious virus-like particles (VLPs) that mimic the viral capsid show an attractive possibility of presenting immunogenic epitopes or targeting molecules on their surface. Here, functionalization of norovirus-derived VLPs by simple non-covalent conjugation of various molecules is shown. By using the affinity between a surface-exposed polyhistidine-tag and multivalent tris-nitrilotriacetic acid (trisNTA), fluorescent dye molecules and streptavidin-biotin conjugated to trisNTA are displayed on the VLPs to demonstrate the use of these VLPs as easily modifiable nanocarriers as well as a versatile vaccine platform. The VLPs are able to enter and deliver surface-displayed fluorescent dye into HEK293T cells via a surface-attached cell internalization peptide (VSV-G). The ease of manufacturing, the robust structure of these VLPs, and the straightforward conjugation provide a technology, which can be adapted to various applications in biomedicine.
Assuntos
Biotecnologia/métodos , Portadores de Fármacos/química , Norovirus/imunologia , Vacinas de Partículas Semelhantes a Vírus/química , Vacinas Virais , Animais , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Peptídeos Penetradores de Células/química , Epitopos/genética , Epitopos/imunologia , Células HEK293 , Histidina/química , Humanos , Ácido Nitrilotriacético/química , Norovirus/genética , Células Sf9 , Tecnologia Farmacêutica/métodos , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologiaRESUMO
Surfaces capable of high-affinity binding of biomolecules are required in several biotechnological applications, such as purification, transfection, and sensing. Therein, the rod-shaped, colloidal cellulose nanocrystals (CNCs) are appealing due to their large surface area available for functionalization. In order to exploit electrostatic binding, their intrinsically anionic surfaces have to be cationized as biological supramolecules are predominantly anionic. Here we present a facile way to prepare cationic CNCs by surface-initiated atom-transfer radical polymerization of poly(N,N-dimethylaminoethyl methacrylate) and subsequent quaternization of the polymer pendant amino groups. The cationic polymer brush-modified CNCs maintained excellent dispersibility and colloidal stability in water and showed a ζ-potential of +38 mV. Dynamic light scattering and electron microscopy showed that the modified CNCs electrostatically bind cowpea chlorotic mottle virus and norovirus-like particles with high affinity. Addition of only a few weight percent of the modified CNCs in water dispersions sufficed to fully bind the virus capsids to form micrometer-sized assemblies. This enabled the concentration and extraction of the virus particles from solution by low-speed centrifugation. These results show the feasibility of the modified CNCs in virus binding and concentrating, and pave the way for their use as transduction enhancers for viral delivery applications.
Assuntos
Bromovirus , Celulose/química , Coloides/química , Norovirus , Polímeros/química , Animais , Ânions , Brometos/química , Capsídeo , Cátions , Linhagem Celular , Hidrocarbonetos Iodados/química , Insetos , Luz , Espectroscopia de Ressonância Magnética , Metacrilatos/química , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Nanopartículas/química , Norovirus/metabolismo , Nylons/química , Ligação Proteica , Espalhamento de Radiação , Espectroscopia de Infravermelho com Transformada de Fourier , Eletricidade Estática , Propriedades de SuperfícieRESUMO
Coxsackievirus B3 (CVB3) is an important cause of acute and chronic viral myocarditis, and dilated cardiomyopathy (DCM). Although vaccination against CVB3 could significantly reduce the incidence of serious or fatal viral myocarditis and various other diseases associated with CVB3 infection, there is currently no vaccine or therapeutic reagent in clinical use. In this study, we contributed towards the development of a CVB3 vaccine by establishing an efficient and scalable ion exchange chromatography-based purification method for CVB3 virus and baculovirus-insect cell-expressed CVB3 virus-like particles (VLPs). This purification system is especially relevant for vaccine development and production on an industrial scale. The produced VLPs were characterized using a number of biophysical methods and exhibited excellent quality and high purity. Immunization of mice with VLPs elicited a strong immune response, demonstrating the excellent vaccine potential of these VLPs.
Assuntos
Infecções por Coxsackievirus/imunologia , Enterovirus Humano B/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Animais , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos , Baculoviridae/genética , Cromatografia por Troca Iônica , Infecções por Coxsackievirus/prevenção & controle , Modelos Animais de Doenças , Feminino , Ordem dos Genes , Vetores Genéticos/genética , Imunidade Celular , Imunização , Camundongos , Vacinas de Partículas Semelhantes a Vírus/isolamento & purificação , Vacinas de Partículas Semelhantes a Vírus/ultraestruturaRESUMO
Recombinant expression of the norovirus capsid protein VP1 leads to self-assembly of non-infectious virus-like particles (VLPs), which are recognized as promising vaccine candidates against norovirus infections. To overcome the scalability issues connected to the ultracentrifugation-based purification strategies used in previous studies, an anion exchange-based purification method for norovirus VLPs was developed in this study. The method consists of precipitation by polyethylene glycol (PEG) and a single anion exchange chromatography step for purifying baculovirus-expressed GII.4 norovirus VLPs, which can be performed within one day. High product purity was obtained using chromatography. The purified material also contained fully assembled monodispersed VLPs, which were recognized by human sera containing polyclonal antibodies against norovirus GII.4.
Assuntos
Cromatografia por Troca Iônica/métodos , Norovirus/genética , Virologia/métodos , Virossomos/genética , Virossomos/isolamento & purificação , Baculoviridae , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Precipitação Fracionada , Vetores Genéticos , Polietilenoglicóis/químicaRESUMO
Noroviruses are an important cause of epidemic acute gastroenteritis in humans. In this study the production and characterization of GII.4 norovirus virus-like particles (VLPs) in insect cells is reported. Furthermore, the expression of corresponding norovirus polyhistidine-tagged P domain protein in Escherichia coli is described. The protruding P domain of the norovirus capsid is known to contain determinants for antibody and receptor binding. Therefore, P domain proteins were studied as an alternative diagnostic tool for evaluating norovirus infection. Analyses by dynamic light scattering and cryo-electron microscopy revealed the presence of intact VLPs with an average diameter of about 40 nm. Immunostaining and ELISA assays using norovirus-specific human sera revealed that VLPs and the P domain are recognized by norovirus-specific antibodies and by their putative receptor. The VLPs and P domain protein are potentially useful in the development of diagnostic and vaccination tools for noroviruses.
Assuntos
Norovirus/genética , Norovirus/imunologia , Proteínas Virais/genética , Proteínas Virais/imunologia , Virossomos/imunologia , Virossomos/isolamento & purificação , Animais , Anticorpos Antivirais/sangue , Infecções por Caliciviridae/diagnóstico , Infecções por Caliciviridae/prevenção & controle , Linhagem Celular , Escherichia coli/genética , Expressão Gênica , Humanos , Imunoensaio , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Spodoptera , Vacinas Virais/imunologia , Virossomos/genética , Virossomos/metabolismoRESUMO
Norovirus (NoV) -derived virus-like particles (VLPs) resemble empty shells of the virus and NoV P-particles contain only protruding domains of the NoV capsid. Both NoV-derived subviral particles show similar functionality and antigenicity in vitro and are considered to be potential vaccine candidates against NoV gastroenteritis. BALB/c mice were immunized with baculovirus-produced GII-4 VLPs or the corresponding Escherichia coli-produced P-particles by the intramuscular or intradermal route and the NoV-specific antibody and T-cell immune responses were compared. Elevated antibody levels were induced with a single VLP immunization, whereas P-particle immunization required a boost. High avidity antibodies were raised only by VLP immunization. VLP immunization resulted in a balanced T helper type 1/type 2 immune response whereas P-particles induced a T helper type 2-biased response. Only VLP immunization primed T cells for interferon-γ production. Most importantly, cross-reactive B and T cells were induced solely by VLP immunization. In addition, VLP antiserum blocked the binding of heterotypic VLPs to human histo-blood group antigen receptor and saliva. The findings in this study are relevant for the development of NoV vaccines.