Fiber modifications enable fowl adenovirus 4 vectors to transduce human cells

Abstract

Background: Pre-existing immunities hamper the application of human adenovirus (HAdV) vectors in gene therapy or vaccine development. Fowl adenovirus (FAdV)based vector might represent an alternative. Methods: An intermediate plasmid containing FAdV-4 fiber genes, pMD-FAV4Fs, was separated from FAdV-4 adenoviral plasmid pKFAV4GFP. An overlap extension polymerase chain reaction (PCR) was employed for fiber modification in pMD-FAV4Fs, and the modified fibers were restored to generate new adenoviral plasmids through restriction-assembly. FAdV-4 vectors were rescued and amplified in chicken LMH cells. Fluorescence microscopy and flow cytometry were used to evaluate the gene transfer efficiency. The amount of viruses binding to cells was determined by a real-time PCR. A plaque-forming assay and one-step growth curve were used to evaluate virus growth. Results: Four sites in the CD-, DE-, HI-and IJ-loop of fiber1 knob could tolerate the insertion of exogenous peptide. The insertion of RGD4C peptide in the fiber1 knob significantly promoted FAdV-4 transduction to human adherent cells such as 293, A549 and HEp-2, and the insertion to the IJ-loop demonstrated the best performance. The replacement of the fiber2 knob of FAdV-4 with that of HAdV-35 improved the gene transfer to human suspension cells such as Jurkat, K562 and U937. Fiber-modified FAdV-4 vectors could transduce approximately 80% human cells at an acceptable multiplicity of infection. Enhanced gene transfer mainly resulted from increased virus binding. Fiber modifications did not significantly influence the growth of recombinant FAdV-4 in packaging cells.Conclusions: As a proof of principle, it was feasible to enhance gene transduction of FAdV-4 vectors to human cells by modifying the fibers.

CELO Fiber1 Knob Is a Promising Candidate to Modify the Tropism of Adenoviral Vectors

“…Original FAdV-4 vectors could hardly transduce human cells. When FAdV4-CG, a recombinant FAdV-4 carrying GFP reporter gene, was used at an MOI of 1000 vp/cell, less than 4% of 293 cells were detected to express GFP [ 21 ]. In order to increase the transduction of FAdV-4 to human cells, RGD4C peptide was inserted to the CD, DE, HI and IJ loops of FAdV-4 fiber1 knob to generate fiber-modified viruses in a previous study [ 21 ].…”

“…The gene transfer ability of FAdV-4 vector to human cells was very low. We tried to insert RGD4C peptide to the fiber1 knob and replace the knob of fiber2 with that from HAdV-35 fiber, such modification substantially improved the gene transduction of FAdV-4 to human adherent or suspension cell lines [ 21 , 22 , 23 ]. However, the activity of current FAdV-4 vectors still did not meet the requirement of transducing human cells.…”

A Novel Fiber-1-Edited and Highly Attenuated Recombinant Serotype 4 Fowl Adenovirus Confers Efficient Protection Against Lethal Challenge

“…Whether the recombinant virus FAdV4-RFP_F1 with the edited fiber-1 can alter the tissue tropism in vivo needs to be elucidated. Recently, although Zhang et al showed that the knob of Fiber-1 could also be edited to insert the exogenous peptide, several generated recombinant viruses could alter the cell tropism in vitro ( 18 ). To investigate the mechanism for the protective efficacy for FAdV4-RFP_F1 against FAdV-4, the sera from chickens before challenge were collected and tested for neutralizing antibody.…”

No Genus-Specific Gene Is Essential for the Replication of Fowl Adenovirus 4 in Chicken LMH Cells

“…Mastadenoviruses, especially human adenovirus C (HAdV-C), have been intensively studied and constructed as gene transfer vectors ( 2 – 4 ). The application of HAdV-based vectors in human gene therapy and vaccine development has been hindered by high prevalence of pre-existing immunities against these pathogens in humans ( 5 , 6 ), and interest has been attracted to construct recombinant fowl adenoviruses (FAdVs), a subgroup of aviadenoviruses, as gene delivery tools ( 7 – 9 ).…”