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1.
J Pharm Sci ; 110(10): 3375-3384, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34186069

RESUMO

During the manufacturing of recombinant adeno-associated virus vectors, it is generally difficult to purify out vectors that lack nucleic acids (empty particles, EPs), contain incomplete nucleic acids (intermediate particles, IPs) or aggregates. These impurities may cause side effects and therefore it is essential to both quantify and reduce them; however, comprehensive identification of the size distribution and components of virus vectors have been lagging. We developed multiwavelength sedimentation velocity analytical ultracentrifugation to characterize EPs, full particles, IPs, and aggregates in adeno-associated virus vector samples. The wavelength-dependent ultraviolet (UV) absorption of capsid protein and encapsulated single-stranded DNA could be deduced from the multiwavelength detection followed by size distribution analysis and peak area integration. Subsequently, a spectral deconvolution analysis using the wavelength-dependent UV absorption data enabled the identification of the protein-nucleic acid ratio of all species. A comprehensive approach for quantifying the viral vector particles and related impurities was established.


Assuntos
Dependovirus , Vetores Genéticos , Proteínas do Capsídeo , Dependovirus/genética , Ultracentrifugação , Vírion
2.
PLoS One ; 16(1): e0245244, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33412571

RESUMO

IgA antibodies, which are secreted onto the mucosal surface as secretory IgA antibodies (SIgAs), play an important role in preventing influenza virus infection. A recent study reported that anti-hemagglutinin (HA) head-targeting antibodies increase anti-viral functions such as hemagglutination inhibition (HI) and virus neutralization (NT), in addition to HA binding activity (reactivity) via IgA polymerization. However, the functional properties of anti-viral IgA antibodies with mechanisms of action distinct from those of anti-HA head-targeting antibodies remain elusive. Here, we characterized the functional properties of IgG, monomeric IgA, and polymeric IgA anti-HA stalk-binding clones F11 and FI6, and B12 (a low affinity anti-HA stalk clone), as well as Fab-deficient (ΔFab) IgA antibodies. We found that IgA polymerization impacts the functional properties of anti-HA stalk antibodies. Unlike anti-HA head antibodies, the anti-viral functions of anti-HA stalk antibodies were not simply enhanced by IgA polymerization. The data suggest that two modes of binding (Fab paratope-mediated binding to the HA stalk, and IgA Fc glycan-mediated binding to the HA receptor binding site (RBS)) occur during interaction between anti-stalk HA IgA antibodies and HA. In situations where Fab paratope-mediated binding to the HA stalk exceeded IgA Fc glycan-mediated binding to HA RBS, IgA polymerization increased anti-viral functions. By contrast, when IgA Fc glycan-mediated binding to the HA RBS was dominant, anti-viral activity will fall upon IgA polymerization. In summary, the results suggest that coordination between these two independent binding modules determines whether IgA polymerization has a negative or positive effect on the anti-viral functions of anti-HA stalk IgA antibodies.


Assuntos
Hemaglutininas , Imunoglobulina A , Vacinas contra Influenza , Influenza Humana , Animais , Cães , Feminino , Humanos , Camundongos , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Afinidade de Anticorpos , Sítios de Ligação de Anticorpos , Células Cultivadas , Células HEK293 , Hemaglutininas/química , Hemaglutininas/imunologia , Imunogenicidade da Vacina , Imunoglobulina A/química , Imunoglobulina A/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Células Madin Darby de Rim Canino , Camundongos Endogâmicos BALB C
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