Funtional and applied studies on the
adenovirus minor capsid protein IX
Vellinga, J.
Citation
Vellinga, J. (2006, June 29). Funtional and applied
studies on the adenovirus minor capsid protein IX.
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Efficient Incorporation of a Functional Hyper Stable Single-Chain
Antibody Fragment Protein-IX fusion in the Adenovirus Capsid
Jort Vellinga1, Jeroen de Vrij1, Taco Uil1, Diana J.M. van den Wollenberg1, Steve J. Cramer1, Martijn J.W.E. Rabelink1, Leif Lindholm2 and Rob C. Hoeben1
1Department of Molecular Cell Biology, Leiden University Medical Centre, Wassenaarseweg
72, 2333 AL Leiden, The Netherlands
2Got-a-Gene AB, Stena Center 1B, Gothenburg, Sweden
Correspondence: Rob C. Hoeben; Virus Biology Laboratory, Department of Molecular Cell Biology, Leiden University Medical Centre, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands
Summary Recombinant adenoviruses are frequently used as gene transfer vehicles for human
prophylactic and therapeutic gene delivery. Strategies to amend their tropism include the incorporation of polypeptides with high affinity for cellular receptors on the target cell surface. Single-chain antibodies have a great potential to achieve such cell type-specificity. In this study we evaluated the efficiency of incorporation of a single-chain antibody genetically fused with the adenovirus minor capsid protein IX in the capsid of adenovirus type 5 vectors. To this end a hybrid gene was constructed in which the codons for the single-chain antibodies scFv 13R4 were fused with the region coding for the C-terminus of the pIX via a 75-Angstrom spacer sequence. The 13R4 is a hyper-stable single-chain antibody directed against E-galactosidase, which was selected for its
capacity to fold correctly in a reducing environment such as the cytoplasm. A lentiviral vector was used to stably express the pIX.flag.75.13R4.MYC.HIS fusion gene in 911 helper cells. Upon propagation of pIX-gene deleted HAdV-5 vectors on these helper cells the pIX-fusion protein was efficiently incorporated in the capsid. Here, the 13R4 scFv was functional as was evident from its capacity to bind its ligand E-galactosidase on the
outside of the virions. These data demonstrate that the minor capsid protein IX can be used as an anchor for incorporation of single-chain antibodies in the capsids of adenovirus vectors.
Introduction
Human Adenovirus (HAdV)-derived vectors are among the most frequently used gene delivery vehicles for human gene therapy and vaccination. Much effort has been devoted to improve the cell-type specificity of gene delivery. Whereas the use of bispecific antibodies has been employed with considerable success, retargeting of adenovirus vectors by genetic incorporation of cell specific-ligands and single-chain antibodies proved more difficult. Many attractive ligands for
their function1,3. The reducing environment in the cytoplasm, which prevents the formation of disulphide bridges, and the absence of accessory factors to help these proteins to fold correctly, avert their maturation to functional proteins4. Indeed, studies in scFv were fused with capsid proteins were not very successful1,5,6. However, some antibodies can be produced in a soluble form in the cytoplasm and retain their activity. These are the called hyper-stable single-chain antibodies7-11. Recently, such hyper-stable scFv have been incorporated in HAdV particles on de-knobbed, fibritin-foldon trimerized fibers3. Here we demonstrate efficient and functional incorporation of the hyper-stable scFv 13R4 that was fused with the minor capsid protein pIX via a 75-Angstrom spacer. The scFv 13R4 originates from a naïve human phage display library12 and was isolated after random mutagenesis by error-prone polymerase chain reaction, and selection for increased cytoplasmic solubility. We show that 13R4 fused with pIX via the 75-Angstrom spacer is accessible on the surface of purified viruses. Moreover the 13R4 is functional in the capsid as evidenced from its capacity to bind E.coli E-galactosidase.
Methods Cells
The HAdV-5 E1-transformed cell line 91113 was maintained at 37oC in a humidified atmosphere of 5% CO2 in DMEM medium (Gibco-BRL, Breda, The Netherlands) supplemented with 8% fetal bovine serum (Gibco-BRL) and 0.3 % glucose (J.T.Baker, Deventer, The Netherlands). The 911 cells were used to propagate and titer adenovirus vectors. Infections of the cells with HAdVs were carried out in infection medium containing 2% horse serum.
Production of recombinant lentiviruses
The lentiviral vectors used in this study were described in earlier studies14. The lentivirus vectors were derived from the plasmid CMV-eGFP. Plasmids pLV- CMV-pIX.flag.75.13R4.MYC.HIS-IRES-NPTII and pLV-CMV-HissFv.rec-IRES-NPTII, have been constructed by standard cloning procedures14. The gene for pIX.flag.75 was obtained from the pCDNA3.1-based construct pAd5pIX.MYC.flag.75.MYC15.The gene encoding the scFv 13R4 was subcloned from the plasmid pPM163R48. The gene for HissFv.rec was obtained from Dr. David Curiel16. The lentiviral vectors were produced and quantified as described previously14,17. For titer estimations is was assumed that 1 ng p24 is equal to 2 x 103 transducing units in an infection assay18.
Lentiviral transduction
For transduction, the lentiviral supernatant was added to fresh medium together with 8 Pg/ml Polybrene (Sigma Aldrich, Zwijndrecht, The Netherlands). After overnight incubation, the medium was replaced with fresh medium. Cells transduced with lentiviral vectors containing the neomycine selection gene were cultured in medium supplemented with 200 mg.l-1 G418 (Invitrogen, Breda, The Netherlands).
Adenovirus vectors
The CMV.GFP/LUC and HAdV-5'pIX.CMV.GFP/LUC were made as described previously15. The vectors carry a green fluorescent protein (GFP) and a firefly luciferase (LUC) transgene, each under the control of the human cytomegalovirus (CMV) immediate-early promoter. HAdV-5 was obtained from the virus collection of the Dept of Molecular Cell Biology of the Leiden University Medical Center. The infectious titer of the adenovirus vectors was determined by Efficient Incorporation of a Functional Hyper Stable Single-Chain
Antibody Fragment Protein-IX fusion in the Adenovirus Capsid
plaque assay13. For the retargeting assay, 911 cells and 911-HissFv.rec cells were infected with HAdV-5'pIX.CMV.GFP/LUC viruses loaded with pIX.flag.75.13R4.MYC.HIS with a multiplicity of infection (MOI) of 1 or 10. After 24 h, the infection efficiencies were determined by measuring the luciferase activity as described before19.
Western analysis
Cell lysates were made in RIPA lysis buffer (50 mM Tris.Cl pH=7.5, 150 mM NaCl, 0.1% SDS, 0.5% DOC and 1% NP40). Protein concentrations were measured via the standard method with the BCA protein assay (Pierce, Perbio Science B.V., Etten-Leur, The Netherlands). Virus lysates were prepared by adding 5x109 virus particles (measured by a standard OD260 protocol20) directly to Western sample buffer. The Western blotting and
detection procedures have been previously described14,15.
FIG. 1. Schematic representation of the lentiviral system. The lentiviral vectors used in this study are so-called SIN vectors23
, and contain the Rev-responsive element sequence24
, the central poly-purine tract (cPPT)18,25,26, and the human hepatitis B virus-derived posttranscriptional regulatory element. The encephalomyocarditis virus internal ribosomal entry site (IRES) was obtained from pTM327
, the NPTII coding region was isolated from peGFPn2 (Clontech, BD Biosciences, The Netherlands).
Immunohistochemistry assays
using propidium iodide. Subsequently, the cells were washed and mounted on object glasses using Dabco/Glycerol (Glycerol, 0.02M Tris.Cl pH=8.0 and 1 ȝg/ml 2.4-diamidino-2-phenylindole). Cells were visualized with a Leica DM-IRBE microscope.
E-galactosidase binding assay Purified viruses (5 x 109 particles per well) were trapped together with E-galactosidase (Sigma Aldrich, Zwijndrecht, The Netherlands) on DAKO IDEIA microwell strips (Dako Ltd, Ely, United Kingdom). After 60 minutes incubation on room temperature, the microwell strips were washed 8 times according to the IDEIA kit instructions. After adding 60 Pl ONPG (stock was 4 mg/ml in sodium-phosphate buffer, pH7.5) (Sigma Aldrich, Zwijndrecht, The Netherlands) and 60 Pl Z-buffer (100 mM NaH2PO4/Na2HPO4pH 7.5; 10 mM KCl; 1 mM MgSO4 and 50 mM 2-mercaptoethanol) the reaction was stopped after 60 minutes by adding 50 Pl Na2CO3 (stock was 1 M Na2CO3). The conversion of ONPG as a measurement for B-galactosidase activity was determined by assaying the OD420.
Results
For incorporation of the 13R4 scFv in the adenovirus capsid a fusion gene was constructed in which the coding region of pIX was fused via the flag tag with the codons for a 75Angstrom spacer, and with the codons for the 13R4 scFv. The resulting fusion gene coding for pIX.flag.75.13R4.MYC.HIS was inserted into the lentiviral expression vector pLV-CMV-IRES-NPTII14. A schematic outline of the vector is provided in Fig. 1. To test the pIX.flag.75.13R4.MYC.HIS production after lentivirus transduction, 911 cells were exposed to LV-CMV-pIX.flag.75.13R4.MYC.HIS-IRES-NPTII
at 40 ng p24 per 105 cells (911-pIX.flag.75.13R4.MYC.HIS). Forty-eight hours post transduction, the 911-pIX.flag.75.13R4.MYC.HIS cells were fed fresh medium with 200 ȝg/ml G418. No clonal cells were isolated, since the lentiviral transduction leads to a polyclonal cell line that produce homogenous levels pIX, sufficient to fully restore the heat stable phenotype of the HAdV particle14. Immunohistochemistry analysis showed homogeneous amounts of the fusion proteins in the transduced cells (Fig. 2A). The pIX.flag.75.13R4.MYC.HIS amounts produced by the transduced 911 cells are similar to the pIX level produced by 911 cells during infection with a wild-type (wt) HAdV-514(Fig. 2B).
Next, we tested the incorporation of pIX.flag.75.13R4.MYC.HIS into the capsid of the 5 vector HAdV-5'pIX.CMV.GFP/LUC14. This vector lacks a functional pIX gene and carries the eGFP and the firefly luciferase reporter genes under control of two separate CMV-promoters. HAdV-5'pIX.CMV.GFP/LUC viruses were propagated on the 911-pIX.flag.75.13R4.MYC.HIS cell line, harvested and purified via the conventional CsCl purification method. During purification, particle-associated pIX molecules were separated from the non-associated pIX, since variants of pIX that cannot be incorporated into the capsid do not co-purify with the particles of CsCl gradients15,21.To examine the amount of pIX.flag.75.13R4.MYC.HIS in the particles, 5x109 CsCl-gradient purified particles were analyzed by Western analysis (Fig. 2C). The amount of pIX.flag.75.13R4.MYC.HIS in the pIX.flag.75.13R4.MYC.HIS-loaded HAdV-5'pIX.CMV.GFP/LUC particles is similar to the amounts in wt HAdV-5 particles.
Efficient Incorporation of a Functional Hyper Stable Single-Chain Antibody Fragment Protein-IX fusion in the Adenovirus Capsid
To study the functionality of the scFv in the capsid we assayed the ligand (E-galactosidase)-binding capability of the 13R4 on the surface of the adenovirus. To this end, the pIX.flag.75-.13R4.MYC.HIS-loaded HAdV-5'pIX.CMV.GFP/LUC particles were trapped in DAKO IDEIA microwells and incubated with E-galactosidase. The DAKO kit was developed for demonstration of Ad in clinical specimens. It contains microwell strips pre-coated with an anti Ad antibody. After blocking and washing, the particles were exposed to the substrate (ONPG). Compared to wt HAdV-5, the particles that contain the pIX.flag.75.13R4.MYC.HIS molecules showed significant
E-galactosidase activity (Fig. 3). This demonstrates that the 13R4 scFv can bind theE-galactosidase epitope on the surface of the virions.
FIG. 2. A. Immunohistochemistry assay for detection of the pIX.flag.75.13R4 produced by the 911- pIX.flag.75.13R4 cells. The production of pIX.flag.75. 13R4 was visualized using mouse anti-flag and FITC-labeled goat-anti-mouse antibodies. The nuclei were stained using propidium iodide. B. Western analysis. pIX.flag.75.13R4.HIS.MYC levels in the 911-pIX.flag.75.13R4.HIS.MYC cells. The pIX.flag.75.13R4.HIS.MYC amount in the complementing cell line 911-pIX.flag.75.13R4.HIS.MYC was compared with the pIX amounts during wt HAdV-5 infection. The Western analysis was performed using anti pIX serum14. C. Western analysis. Incorporation efficiency of pIX.flag.75.13R4.HIS.MYC. To test the incorporation efficiency of pIX.flag.75.13R4.HIS.MYC produced by the 911-pIX.flag.75.13R4.HIS.MYC cells, HAdV-5'pIX.CMV.GFP/LUC was propagated on the cell line, purified by CsCl centrifugation, and protein lysate of the purified viruses sample was made for Western analysis. The amount of pIX in HAdV-5'pIX.CMV.GFP/LUC propagated on 911-pIX.flag.75.13R4.HIS.MYC was compared with wt HAdV-5, with anti-pIX serum and, as a virus-particle loading control, the 4D2 antibody directed against the fiber protein.
(911-HissFv.rec) produced the HIS receptor, as visualized by the FITC signal from the secondary antibody, whereas the untransduced 911 cells did not (Fig. 4A). To assay the retargeting efficiency of the HAdV-5'pIX.CMV.GFP/LUC loaded with pIX.flag.75.13R4.MYC.HIS, 911 cells and 911-HissFv.rec cells were infected with a multiplicity of infection (MOI) of 1 and 10. To distinguish between infection mediated by the HIS-receptor and infection via the CAR receptor, the viruses were pre-incubated with HIS, anti-fiber-knob (1D6.14) or a mixture of both anti-HIS and anti-fiber-knob. The infection efficiency was measured by means of luciferase transgene delivery 24 hours post infection (Fig. 4B). Pre-incubation with anti-HIS did not result in a reduction of the infection efficiency on both 911 cells and 911-HissFv.rec cells. The slight increased infection efficiency showed with the samples that were pre-incubated with anti-HIS was not significant. The native route of infection of 911 cells is mediated by binding of the viral fiber-knobs to the cellular CAR receptor. This was shown by the inhibition of the infection efficiency after pre-incubation with anti-fiber-knob. To reveal whether the HIS-tag could mediate virus infection via the HIS-receptor on the 911-HissFv.rec cells, the virus was pre-incubated with both anti-fiber-knob and anti-HIS. There was no increased reduction of infection caused by the anti-HIS in addition to the inhibiting activity of the anti-fiber-knob. The results obtained with HAdV-5'pIX.CMV.GFP/LUC loaded with pIX.flag.75.13R4.MYC.HIS were similar on both cell lines, 911 and 911-HissFv.rec. From these results it can be concluded that there is no evidence for retargeting mediated by the HIS-tag on the C-terminus of the pIX.flag.75.13R4.MYC.HIS fusion protein.
Discussion
FIG. 3.E-galactosidase bindings assay. To measure the binding capability of 13R4 on the surface of the virion to its native ligand E-galactosidase, the HAdV-5'pIX.CMV.GFP/LUC loaded with pIX.flag.75.13R4.MYC.HIS were incubated withE-galactosidase and trapped on a DAKO IDEIA strips. The bound E-galactosidase was detected by measuring OD420 after adding ONPG together with the Z-buffer (n=3). As negative control wt HAdV-5 was used.
Here we demonstrate the functional incorporation of a hyper-stable scFv (13R4), directed against E-galactosidase, fused with pIX into the adenovirus capsid. To test whether the pIX.flag.75.13R4.MYC.HIS (51.8 kDa) is able to incorporate into the viral capsid a 911 cell line was created that produced the pIX.flag.75.13R4.MYC.HIS proteins. The cell line was created by transduction of 911 cells with LV-CMV-pIX.flag.75.13R4.MYC.HIS-IRES-NPTII. The production of pIX.flag.75.13R4.MYC.HIS proteins in the 911 cells was homogeneous as was seen earlier with pIX and pIX.flag.75.MYC proteins14. Propagation of HAdV-5'pIX.CMV.GFP/LUC on the Efficient Incorporation of a Functional Hyper Stable Single-Chain
Antibody Fragment Protein-IX fusion in the Adenovirus Capsid
911- pIX.flag.75.13R4.MYC.HIS cells did result in virus production levels that are similar to the levels obtained after propagation on the standard 911 helper cell line. Western analysis showed that pIX.flag.75.13R4.MYC.HIS was incorporated into the HAdV-5'pIX.CMV.GFP/LUC as efficient as wt pIX (14.3 kDa) in wt HAdV-5 particles. This demonstrates that insertions of ligands up to 2.5 times the size of wt pIX can incorporated into the capsid without decreasing the incorporation efficiency. TheE-galactosidase assay demonstrated that the 13R4 fusion molecule is able to bind its target (E-galactosidase) on the outer surface of the virus capsid. The capacity to bind E-galactosidase was
specific for the particles harboring the pIX.flag.75.13R4.MYC.HIS molecules since HAdV-5'pIX showed minute enzyme activity. This is the first time that it has been shown that pIX can be used as anchor to incorporate large targeting ligands such as the model scFv 13R4 into the HAdV-5 capsid.
Unfortunately, the E-galactosidase is not available as receptor on cell surfaces. This makes it difficult to formally show that the scFv can mediate adenovirus retargeting. To reveal whether the pIX.flag.75.13R4.MYC.HIS was able to mediate infection of HAdV-5'pIX.CMV.GFP/LUC loaded with pIX.flag.75.13R4.MYC.HIS we used 911 cells and HissFv.rec cells. The
HissFv.rec cells produced an artificial receptor, specific for HIS-tagged proteins that is presented on the membrane surfaces of these cells. Unfortunately, this study did not reveal evidence for retargeting via the C-terminal HIS tag of the pIX.flag.75.13R4.MYC.HIS molecules. It should be noticed that it was not known whether the C-terminal HIS-tag was accessible for cellular receptors. Therefore the results from the retargeting experiment were not conclusive. Further research on the accessibility of the C-terminal HIS-tag might shine some light on this aspect. Furthermore, the strategy that was used in this study to block the native binding of the HAdV-5 fiber to CAR by antibodies directed against the knob of the fiber could have a spatial hindrance upon the pIX.flag.75.13R4.MYC.HIS mediated retargeting15,22. Blockage of the fiber-knob by antibodies as was done in this study could even induce a stronger spatial hindrance upon pIX-fusion protein mediated retargeting. In this regard, the future studies involving fiberless HAdV might be useful to elucidate whether pIX-fusion proteins can mediate cell targeting. Studies on scFv improved the understanding between structure and function of these proteins and resulted in scFv that can be produced without the need of stabilizing disulfide bounds. The development of techniques that facilitate the creation of large libraries of hyper stable scFv will be of great importance for use in therapeutic agents such as gene transfer vectors described in this study. Traditional techniques such as isolation of VH and VL domains to construct scFv from an original hybridoma or in vitro display systems to screen and select for specific scFv that consequently should be tested for their ability to fold into functional scFv in the cytoplasm. However, these techniques have been proven to work for standard scFv they result in low yields of the hyper
stable scFv variants. Other techniques such as intracellular antigen capturing (IAC) and CDR grafting are promising approaches to create effective hyper stable scFv that can be used for retargeting of adenoviruses to specific cells or tissues. The efficient incorporation of the relatively large 13R4.MYC.HIS fusion protein is promising for future retargeting strategies. The fact that the model scFv used in this study is biological active on the surface of the adenovirus is stimulating for insertion of scFv that are directed against specific cellular receptors. Further studies should reveal whether this strategy can lead to specific adenovirus retargeting.
Acknowledgements
We thank Dr. David Curiel for supplying the 1D6.14 antibody and the plasmid containing the HissFv.rec gene. This work was supported by the Technology Foundation STW (program LGN66.3977), and the European Union through the 6th Framework program GIANT (Contract no.: 512087).
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Efficient Incorporation of a Functional Hyper Stable Single-Chain Antibody Fragment Protein-IX fusion in the Adenovirus Capsid
