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.


| INTRODUCTION
Adenoviruses are non-enveloped viruses containing linear doublestranded DNA genomes of 26-48 kb. 1 The family Adenoviridae consists of five genera, among which Mastadenoviruses infect mammalian hosts exclusively, whereas Aviadenoviruses have been found only in birds. 2 Human adenoviruses (HAdVs) have been comprehensively studied and are constructed as gene transfer vectors for gene therapy and vaccine development. [3][4][5] Adenoviral vectors have many advantages compared to other viral gene transfer tools: the mediumsized genomes are manipulable and suitable for stably loading transgene up to 7 kb or even longer; they can be produced with a high yield and reasonable cost; and their non-enveloped virions, with physicochemical stability, are convenient for preservation and usage.
Moreover, adenoviruses feature efficient gene transfer to cells at a high multiplicity of infection (MOI) and adenoviral vectors do not integrate into chromosomes, which reduces the risk of causing cellular transformation. However, the pre-existing immunity against HAdV impairs the efficacy and safety of these vectors in clinical practice. 6,7 HAdVs are ubiquitous pathogens and generally cause mild, selflimiting infections in immunocompetent people. 8,9 HAdV-5-based vectors have been commonly used. The seroprevalence of HAdV-5 neutralizing antibody (NAb) varies in different geographic regions and was reported to be 50-90% in healthy adults. [10][11][12] Such a situation has attracted interest regarding the development of vectors based on HAdV serotypes with a lower seroprevalence or on chimpanzee adenoviruses (ChAdVs). [13][14][15][16] Recent epidemiological data demonstrate that geographical distributions of pathogens are related to climate, temporal variation and immigration and are not limited by geopolitical borders. For example, HAdV-D26 is generally considered to have low seroprevalence and HAdV-D26 NAb was detected to be positive in less than 12% of the sera collected from the USA and European countries. However, the HAdV-D26 seroprevalence was 70-90% in some African countries and 35-60% in China and Thailand. 11 Even though non-human primate (NHP) adenoviruses are less likely to infect human, the pre-existing immunity in human was detected as a result of the cross-reactivity between HAdV and ChAdV. 11,17 Therefore, we need to address the pre-existing immunity for clinical application of both HAdV and NHP adenovirus vectors.
Fowl adenoviruses (FAdVs) have the potential to be constructed as vectors targeting human cells, whereas these vectors have low gene transfer efficiency. FAdVs belong to the genus of Aviadenovirus 1 and 12 serotypes (FAdV-1 to -8a and -8b to 11) of FAdV have been identified, which belong to five species (FAdV-A to -E). 18 CELO (chicken embryo lethal orphan) virus is the model type of FAdV-A1 and has been constructed as gene transfer vectors. 19 FAdV vectors have two advantages over HAdV counterparts: a lack of pre-existing immunity against FAdV in human and a low cost of producing FAdV in chicken embryo, although the biosafety of FAdVs has not been studied. It was reported that FAdV-1 could infect mammalian cells by binding to CAR receptors with its fiber1, although doubts were raised by the results of crystal structure studies. 20,21 FAdV-1 vectors, even with a modified fiber1, could only transduce mammalian cells with limited efficiency. 22 Fiber modification can change the tropism of adenoviruses because fiber is the main ligand for virus to bind to its cellular receptor. Arginine-glycine-aspartic acid (RGD) peptide family is known as the most prominent ligand for the extracellular domain of integrin receptors. [23][24][25][26][27] RGD4C peptide can bind to integrin with high affinity and promote adenovirus infection when incorporated into the fiber knob. 4,28,29 However, the incorporation of RGD4C into the receptor binding site of the fiber knob (e.g. the insertion of RGD4C into the HI loop of HAdV-35 fiber knob) would impair the interaction between fiber and its native receptor. 30 We established a vector system based on FAdV-4, and found that fiber1 was the essential gene, whereas fiber2 was dispensable for FAdV-4 infection. 31

| Plasmid construction
We took the insertion of RGD4C CDS to fiber1 CD loop as an example to determine the procedure of fiber1 modification (see Supporting information, Figure S1). With pMD-FAV4Fs as the template, PCR was performed using the primer 1805FAV4F1Mf/1805FAV4F1CDRr to amplify SacI-CDR fragment (119 bp) and the primer 1805FAV4F1CDRr/1805FAV4F1Mr to amplify CDR-AgeI fragment (475 bp). pMD-FAV4Fs was digested with SacI/AgeI, and then the fragment of 5124 bp was recovered after electrophoresis, mixed with the SacI-CDR and CDR-AgeI fragments and subjected into DNA assembly to generate the modified shuttle plasmid pMD-FAV4F1CDR. pMD-FAV4F1CDR was digested with KpnI/EcoRV to recover fragment containing fibers (2997 bp), pKFAV4GFP was digested with MauBI/SbfI to recover the large fragment (43,078 bp) and these two fragments were mixed for DNA assembly to generate adenoviral plasmid pKFAV4F1CDR-GFP (restriction-assembly; see Supporting information, Figure S2). 34,35 To insert RGD4C into the fiber2 CD loop, overlap extension PCR was performed to amplify F1-F2CDR fragment (3007 bp), which was inserted at the MauBI/SbfI sites in pKFAV4GFP to generate pKFAV4F2CDR-GFP through DNA assembly (see Supporting information, Figure S3). The intermediate plasmid-based system was employed for replacing the CMV promoter with human EF1a promoter to generate adenoviral plasmid pKFAV4-EG. 32 pKFAV4-EG was a progeny plasmid of pKFAV4-CX19A and different from pKFAV4-GFP in ORF0 and ORF19A deletion and the replacement of GFP promoter (the detailed procedure will be provided upon request). F1IJR-F2 fragment was amplified by PCR with pKFAV4F1IJR-GFP as the template and primers 1811MD-FAV4FSf/r and inserted at the MauBI/SbfI sites in pKFAV4-EG to generate pKFAV4F1IJR-EG by DNA assembly. The F1IJR-F35K fragment was amplified by overlap extension PCR (Table 1) and inserted at the MauBI/SbfI sites in pKFAV4-EG to generate pKFAV4FIJ35K-EG by DNA assembly.

| Rescue, purification and titration of recombinant viruses
Adenoviral plasmid was linearized by PmeI digestion, recovered and used to transfect LMH cells. GFP focuses could be observed under fluorescence microscope 3 days post transfection. The cells together with culture medium were harvested 2-4 days later, subjected to three freeze-thaw cycles and centrifuged to remove cellular debris. The seed virus was amplified in LMH cells, purified with the traditional ultracentrifugation method except that 10 mM citrate (pH 6.2) instead of 10 mM Tris-Cl (pH 7.6) was used as the buffer solution. 32 Particle titer of purified virus was determined by measuring the content of genomic DNA, where 100 ng of genomic DNA is equivalent to 2.3 Â 10 9 viral particles (vp) because a 43-kb genome has a molecular mass of 2.6 Â 10 7 .
Infectivity titer was determined using LMH cells with the limiting dilution assay by counting GFP+ cells 30 hours post infection. 36

| Statistical analysis
The data are presented as the mean ± SD unless otherwise indicated.
The data collected from one cell line were combined and analyzed with two-way analysis of variance. MOI and virus type were defined F I G U R E 1 Schematic diagram of fiber modification sites, elements and procedure. (A) Amino acid sequence of the FAdV-4 fiber1 knob. The β-strands, which are shown as bold letters and denoted with arrows, were predicted according to the alignment of FAdV fiber knob domains and the crystal structures of FAdV-1 fiber1 (PDB ID 2IUN) and fiber2 (PDB ID 2VTW). 38 The sites for RGD4C insertion are labelled with a solid inverted triangle "▼". 3 | RESULTS

| Construction of adenoviral plasmids
The fiber knob domains of FAdV-1, À4 and À9 have been aligned. 38 After checking the spatial position of every amino acid according to the crystal structures of FAdV-1 fiber1 (PDB ID 2IUN) and fiber2 (PDB ID 2VTW), 20,21 we chose some sites in CD, DE, HI and IJ loops of the fiber1 knob for incorporation of RGD4C peptide ( Figure 1A).
To determine whether fiber2 knob could be similarly modified, we selected the CD loop in fiber2 for RGD4C insertion ( Figure 1B and C).
To increase the transduction efficiency of FAdV-4 to human suspension cells, we replaced the whole knob of fiber2 with that of HAdV-35 ( Figure 1D). Two unique restriction sites of MauBI and SbfI outside the fiber region facilitated the modification. With the help of intermediate plasmid pMD-FAV4Fs, 31 fiber1-modified adenoviral plasmids were constructed ( Figure 1E; see also Supporting information, Figure S1 and S2). Fiber2 or fiber1/fiber2 combined modifications were carried out by DNA assembly of MauBI/SbfI-digested adenoviral plasmid and the product of overlap extension PCR. In the adenoviral plasmids pKFAV4F1IJR-EG and pKFAV4FIJ35K-EG, the promoter of transgene (CMV promoter) was replaced with that of human EF1a gene to increase transgene expression in human suspension cells (Table 2).

| Preparation of recombinant FAdV-4 viruses
After identification with restriction analysis and sequencing of fiber-modified region (Figure 2A, B and C), adenoviral plasmids were linearized and transfected into LMH cells. The growth of GFP foci could be observed under a fluorescence microscope. Enlarged GFP foci formed plaques, and a cytopathic effect occurred as plaques merged ( Figure 2D). Rescued viruses were amplified, purified and titrated. The fiber modification was further confirmed by sequencing the PCR product of the fiber region in the viral genome ( Figure 2E). The structure of the RGD4C-modified fiber knob was predicted with I-TASSER, 39 and the results showed that the RGD4C motif protruded from the original loop, indicating an appropriate selection of insertion sites ( Figure 2F). The information for purified recombinant viruses is summarized in Table 2. For the sake of convenience, short virus names are used in the present study ( Table 2).

| Transduction of adherent cells with RGD4Cincorporated FAdV-4 viruses
Preliminary experiments were performed with 293 and A549 cells to evaluate the gene transduction efficiency of RGD4C-incorporated FAdV-4 viruses ( Figure 3A).  (Figures 3 and 4A, B). Supporting information, Figure S4). It was unexpected that both viruses could transduce HL-60 cells with a high efficiency because previous work showed that HL-60 was inert to the infection of HAdV-5 or fiber-pseudotyped HAdV-5 vectors. 33,42 Both viruses could efficiently transduce Jurkat, K562 and U937 cells ( Figure 4C; see also Supporting information, Figure S4). The efficiency of transduction to K562 or U937 cells was considerably high and the toxicity of FIJ35K-EG caused cell lysis at a MOI of 10,000, which resulted in a decreasing percentage of GFP-positive cells for K562 (see Supporting information, Figure S4). The gene transduction was further investigated with K562 and U937 cells within a restricted MOI range from 1000 to 4000 ( Figure 4D). Although the transduction efficiency of F1IJR-EG increased linearly as the MOIs increased, the highest efficiencies were still low (30% for K562 and 20% for U937).

| The growth of fiber-modified FAdV-4 in packaging cells
The yield of progeny virus needs to be considered for the application of vectors. We first evaluated the plaque-forming abilities of FAdV4-GFP, F1IJR and FIJ35K-EG on LMH cells ( Figure 6A and B).

| DISCUSSION
Humans do not have pre-existing immunity against FAdV, which has led to interest in constructing FAdV vectors for application in humans.
CELO virus (FAdV-A1) was constructed as a gene transfer tool two decades ago. 19 HI loop modification of the fiber1 knob and polymercoating have been employed to improve the transduction of FAdV-A1 to human cells. 22,43 Similar to FAdV-A, FAdV-C also has two types of fiber on the virion. 44 It was reported recently that fiber1 was the major fiber for FAdV-C4 adsorption. 45,46 We obtained the same findings and further confirmed that fiber2 was non-essential for growing FAdV-4 in a cultured chicken cell line, 31 which implies that fiber1 may Besides short peptide insertion, knob substitution is another useful approach for changing the cellular tropism of adenoviruses. 4,28,29 At the very beginning, the sequence encoding fiber1 shaft and knob of FAdV-4 was replaced with that of HAdV-35 in the laboratory.
However, the recombinant virus could not be rescued in LMH cells, which led to the finding that fiber2, instead of fiber1, was dispensable for FAdV-4 propagation. 31 HAdV-35 has a cellular receptor CD46, which is expressed abundantly on human suspension cells. 47 The In conclusion, the present study has identified four sites in the