Adeno-associated viral (AAV) vectors show great promise for gene therapy because of their excellent safety profile; however, development of robust dose-determining assays for AAV has presented a significant challenge. With the ultimate goal of future harmonization and standardization of AAV dose determination assays, we systematically analyzed the influence of key variables, including sample preparation procedure, the choice of primers, and real-time quantitative PCR (qPCR) target sequences and calibration DNA conformation on the qPCR quantitation of AAV products. Our results emphasize the importance of designing qPCR primers and conducting sample preparation and demonstrate the need for extensive characterization, vigorous control, and use of reference materials in clinical dose determination.

During respiratory-virus infection, excessive lymphocyte activation can cause pathology both in acute infection and in exacerbations of chronic respiratory diseases. The costimulatory molecule CD161 is expressed on lymphocyte subsets implicated in promoting respiratory inflammation, including Th2, Th17, mucosally associated invariant T (MAIT) cells, and type 2 innate lymphoid cells. We asked whether the CD161 ligand LLT1 could be expressed on respiratory epithelial cells following respiratory-virus infection as a mechanism by which respiratory-virus infection could promote activation of proinflammatory lymphocytes. In response to respiratory syncytial virus (RSV) infection, expression of LLT1 was upregulated in the BEAS-2B respiratory epithelial cell line and primary human bronchial epithelial cells. Imaging studies revealed that LLT1 expression increased in both RSV-infected and cocultured uninfected cells, suggesting that soluble factors produced during infection stimulate LLT1 expression. TLR3 and TLR2/6 ligands led to a rapid increase in LLT1 mRNA in respiratory epithelial cells, as did the proinflammatory cytokines type I interferons, interleukin 1β (IL-1β), and tumor necrosis factor alpha (TNF-α), which are produced early in respiratory-virus infection. Immunohistochemistry confirmed the increase in LLT1 protein on the epithelial cell surface, and live-cell confocal microscopy demonstrated accumulation of epithelial LLT1 at synapses formed with CD161+ T lymphocytes. LLT1 expression by the respiratory epithelium in response to respiratory-virus infection and inflammatory cytokines represents a novel link between innate immunity and lymphocyte activation. As a regulator of CD161+ proinflammatory lymphocytes, LLT1 could be a novel therapeutic target in inflammation caused by respiratory-virus infection.IMPORTANCE The immune response to respiratory-virus infection is essential for clearing the pathogen but, if excessive, can lead to tissue damage and obstruction of the airways. How viral infection activates immune cells in the lungs is not fully understood. Here, we show that LLT1 can be expressed in lung cells in response to infection. LLT1 triggers CD161, a receptor on inflammatory immune cells. This mechanism may promote activation of immune cells in the lungs in viral infection and could be a novel target for therapies aimed at reducing lung inflammation.

Adeno-associated viral (AAV) vectors show great promise because of their excellent safety profile; however, preexisting immune responses have necessitated the administration of high titer AAV, posing a significant challenge to the advancement of gene therapy involving AAV vectors. Recombinant AAV vectors contain minimum viral proteins necessary for their assembly and gene delivery functions. During the process of AAV assembly and production, AAV vectors acquire, inherently and submissively, various cellular proteins, but the identity of these proteins is poorly characterized. We reason that by identifying host cell proteins inherently associated with AAV vectors we may better understand the contribution of cellular components to AAV vector assembly and, ultimately, may improve the production of AAV vectors for gene therapy. In this study, three serotypes of recombinant AAV, namely AAV2, AAV5, and AAV8, were investigated. We used liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) methods to identify protein composition in purified AAV vectors, confirmed protein identities using western blotting, and explored the potential function of selected proteins in AAV vector production using small hairpin (shRNA) methods. Using LC-MS/MS, we identified 44 AAVassociated cellular proteins including Y-box binding protein (YB1). We showed for the first time that the establishment of a novel producer cell line by introducing an shRNA sequence down-regulating YB1 resulted in up to 45-and 9-fold increase in physical vector genome titers of AAV2 and AAV8, respectively, and up to 7-fold increase in AAV2 transduction vector genome titers. Our results revealed that YB1 gene knockdown promoted AAV2 rep expression and vector DNA production and reduced the number of empty particles in AAV2 products, suggesting that YB1 plays an important role in AAV vector assembly by competition with adenovirus E2A and AAV capsid proteins for binding to the inverted terminal repeat (ITR) sequence. The significance and implications of our findings in future improvement of AAV production are discussed.

Adeno-associated viruses (AAV) contain minimal viral proteins necessary for their replication. During virus assembly, AAV acquire, inherently and submissively, various cellular proteins. Our previous studies identified the association of AAV vectors with the DNA binding protein nucleophosmin (NPM1). Nucleophosmin has been reported to enhance AAV infection by mobilizing AAV capsids into and out of the nucleolus, indicating the importance of NPM1 in the AAV life cycle; however the role of NPM1 in AAV production remains unknown. In this study, we systematically investigated NPM1 function on AAV production using NPM1 knockdown cells and revealing for the first time the presence of G-quadruplex DNA sequences (GQRS) in the AAV genome, the synergistic NPM1-GQRS function in AAV production and the significant enhancement of NPM1 gene knockdown on AAV vector production. Understanding the role of cellular proteins in the AAV life cycle will greatly facilitate high titre production of AAV vectors for clinical use.