A lentiviral vector-based adenovirus fiber-pseudotyping approach for expedited functional assessment of candidate retargeted fibers.
BACKGROUND
Many studies aimed at retargeting adenovirus (Ad) rationally focus on genetic modification of fiber, which is the primary receptor-binding protein of Ad. Retargeted fibers ultimately require functional validation in the viral context.


METHODS
Lentiviral vectors (LV) were used to express fiber variants in cells. Infections with a fiber gene-deleted Ad vector yielded fiber-pseudotyped viruses. An enzyme-linked immunosorbent assay and slot blot-based assays probed target binding-ability of retargeted fibers. Differential treatments with an alkylating agent prior to western blot analysis allowed for examination of intra- and extracellular redox states of fibers.


RESULTS
In the present study, LV-based fiber-pseudotyping of Ad is presented as an accelerated means to test new fibers. LV-mediated gene transfer yielded stable and uniform populations of fiber variant-expressing cells. These populations were found to effectively support fiber-pseudotyping of Ad. As a secondary objective of the study, we functionally assessed a chimeric fiber harboring a tumor antigen-directed single-chain antibody fragment (scFv). This fiber was shown to trimerize and achieve a degree of binding to its antigenic target. However, its capsid incorporation ability was impaired and, moreover, it was unable to confer a detectable level of target binding upon Ad. Importantly, subsequent analyses of this fiber revealed the improper folding of its scFv constituent.


CONCLUSIONS
LV-based fiber-pseudotyping was established as a convenient method for testing modified fibers for functionality within Ad particles. Furthermore, a new chimeric fiber was found to be inadequate for Ad retargeting. The folding difficulties encountered for this particular fiber might be generally inherent to the use (i.e. for genetic Ad capsid incorporation) of complex, disulfide bridge-containing natural ligands.
Uil
Taco G
TG
Leiden University Medical Center, Department of Molecular Cell Biology, Leiden, The Netherlands.
de Vrij
Jeroen
J
Vellinga
Jort
J
Rabelink
Martijn J W E
MJ
Cramer
Steve J
SJ
Chan
On Ying A
OY
Pugnali
Margherita
M
Magnusson
Maria
M
Lindholm
Leif
L
Boulanger
Pierre
P
Hoeben
Rob C
RC
D000256:Adenoviridae / Q000235:genetics* / Q000378:metabolism; D002478:Cells, Cultured; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics* / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology; D014161:Transduction, Genetic
D057127:Single-Chain Antibodies