Expression of a versatile DC-targeting fusion protein using an Adenovirus expression system.
The importance of viral and tumour vaccines in eliciting elicit strong CD8+ T-cell responses has been widely acknowledged. Strategies exploring ways to enhance CD8+ T-cell responses have been developed, including targeting of vaccine antigens to dendritic cell (DC) receptors to access to the cross presentation pathway. Many DC endocytic receptors could potentially lead to augmented CD8+ T-cell responses if antigens were targeted directly to them, however only a few receptors have been explored because current targeting reagents are limited in the number of receptors that they are able to target. Consequently, this study describes the production and purification of a streptavidin-fusion protein that provides a versatile and efficient means to target antigen to more than one DC receptor. A model antigen gene, CMV pp65, and a streptavidin core gene, were spliced together using an overlap-extension PCR technique. The resulting fusion gene was cloned into a vector allowing expression in an Adenovirus-based expression system. Expression was verified and optimised before Ni-NTA affinity chromatography purification. Evaluation of pp65-streptavidin immunogenicity revealed that it elicits similar levels of CD8+ T-cell proliferative responses as pp65 and is able to effectively target specific DC receptors when used in addition to biotinylated receptor-specific antibodies. Additionally, enhancement of CD8+ T-cell responses was shown after directing pp65-strep to selected DC receptors in preliminary in vitro experiments. Collectively, this highlights the ease of production of a streptavidin-fusion protein, and demonstrates its use as a promising strategy to evaluate numerous DC receptors as potential targets in vaccine strategies.
Imperial College London, Immunology Department, Chelsea and Westminster Hospital, London, United Kingdom.
Sirika
S
Pillay
Steven
S
Patterson
D000256:Adenoviridae / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D002460:Cell Line; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D003713:Dendritic Cells / Q000276:immunology*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D006801:Humans; D016133:Polymerase Chain Reaction; D011956:Receptors, Cell Surface / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D019809:Streptavidin / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D013302:Streptomyces / Q000235:genetics / Q000276:immunology; D014765:Viral Vaccines / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification
D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D014765:Viral Vaccines; D019809:Streptavidin