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HUMAN GENE THERAPY 18 413 422 May 2007 Mary Ann Liebert Inc DOI 10 1089 hum 2006 194 Human Immunodeficiency Viral Vector Pseudotyped with the Spike Envelope of Severe Acute Respiratory Syndrome Coronavirus Transduces Human Airway Epithelial Cells and Dendritic Cells GARY P KOBINGER 1 2 MARIA P LIMBERIS 3 SURI SOMANATHAN 3 GREGORY SCHUMER 4 PETER BELL 3 and JAMES M WILSON 3 ABSTRACT The human severe acute respiratory syndrome coronavirus SARS CoV is a highly infectious virus that causes severe respiratory infections in humans The spike envelope glycoprotein of SARS CoV the main determi nant of SARS CoV tropism was isolated and used to pseudotype a human immunodeficiency virus HIV based vector Spike pseudotyped HIV vector was generated and evaluated in vitro on well differentiated hu man airway epithelial cells and bronchial explants and in vivo in murine airways The spike envelope was less efficient at promoting HIV vector transduction of murine airway epithelium than an optimized deletion mu tant of the Zaire ebolavirus envelope glycoprotein NTD6L which was used as a benchmark However spike pseudotyped HIV vector was substantially more efficient than NTD6L pseudotyped vector on human airway epithelium as demonstrated by lacZ gene transfer in primary cultures of epithelial cells and bronchial ex plants In addition this study shows that spike pseudotyped HIV based vector can efficiently transduce hu man dendritic cells and epithelial cells of the esophagus which may have implications in investigating mech anisms of SARS CoV pathogenesis Spike pseudotyped HIV based vector is a novel lung directed gene transfer vehicle that holds promise for the treatment of genetic lung diseases such as cystic fibrosis or H9251 1 antitrypsin deficiency 413 INTRODUCTION L ENTIVIRAL VECTORS can be efficiently pseudotyped with en velope proteins from many viruses including the glyco protein from vesicular stomatitis virus VSV G Naldini et al 1996 Although VSV G pseudotyped lentiviral vectors can transduce many cell types with good efficacy Blomer et al 1997 Miyoshi et al 1997 Li et al 1998 in vivo transduc tion of airway cells was detected only after disruption of ep ithelial integrity Johnson et al 2000 Limberis et al 2002 To achieve good transduction of lung airway epithelia in vivo envelope glycoproteins derived from other viruses have been evaluated in the context of pseudotyped human immunodefi ciency virus HIV based vectors These studies revealed that the envelope glycoprotein of the Zaire ebolavirus can success fully pseudotype HIV based vectors and promote efficient gene transfer to airway epithelia and submucosal glands in vivo Kob inger et al 2001 Medina et al 2003 Additional develop ment of the Zaire ebolavirus led to the generation of two opti mized deletion mutants deletions of the mucin domain NTD4L and NTD6L that further enhanced gene transfer to the airways of mice Medina et al 2003 and nonhuman primates G P Kobinger M P Limberis S Somanathan G Schumer P Bell and J M Wilson unpublished data The severe acute respiratory syndrome coronavirus SARS CoV that emerged in Asia at the end of 2002 had an apparent tropism for the lung based on the fact that infected patients de veloped severe pneumonia Drosten et al 2003 Several stud 1 Special Pathogens Program National Microbiology Laboratory Public Health Agency of Canada Winnipeg MB Canada R3E 3R2 2 Department of Medical Microbiology University of Manitoba Winnipeg MB Canada R3T 2N2 3 Gene Therapy Program Department of Pathology and Laboratory Medicine University of Pennsylvania Philadelphia PA 19104 4 Hydrobiotech 14163 Labelle Mirabel Quebec Canada J7J 1M3 ies indicated that SARS CoV acutely replicated in pulmonary epithelia initiating the cascade of events leading to respiratory distress syndrome and death in approximately 10 of cases Drosten et al 2003 Lee et al 2003 The spike envelope gly coprotein of SARS CoV is the main determinant for virus tro pism and mediates binding to its cellular receptor angiotensin converting enzyme 2 ACE2 receptor Li et al 2003 Hofmann and Pohlmann 2004 Spike glycoprotein was also shown to pseudotype HIV based vector with reasonable efficiency and to mediate transduction of various cell lines in vitro Hofmann et al 2004 Moore et al 2004 Yang et al 2004 The present study evaluates the transduction efficiency of spike pseudotyped HIV based vector in primary cultures of hu man airway epithelial and dendritic cells tracheal explants and in vivo in murine airways after in vivo administration with the goal of delineating SARS pathogenesis and creating better vec tors for lung gene transfer MATERIALS AND METHODS DNA constructs and vector production The helper packaging construct pCMVH9004R8 2 encoding the HIV helper function the transfer vector pHxH11032LacZWP encod ing H9252 galactosidase H9252 Gal Watson et al 2002 and plasmids encoding envelope proteins were used for vector production Plasmid pcDNA NTD6L or pShCAG2 nSpike encoding re spectively the NTD6L or spike viral envelope was used to gen erate pseudotyped HIV vector The generation of plasmid pcDNA NTD6L encoding a deletion mutant of the Zaire ebolavirus envelope glycoprotein has been described previ ously Medina et al 2003 Two variants of the spike enve lope gene of SARS CoV were generated and evaluated with the cytomegalovirus CMV or CMV immediate early enhancer chicken H9252 actin rabbit H9252 globin hybrid CAG promoter by Western blot and vector production The spike envelope gly coprotein complementary DNA cDNA was first isolated from a Canadian isolate of SARS CoV strain Tor2 by reverse tran scriptase polymerase chain reaction RT PCR generating the spike gene The PCR fragment was cloned with a TOPO TA Cloning kit Invitrogen Carlsbad CA and characterized by se quencing performed by SeqWright Houston TX and was found to be 100 identical to the published sequence Marra et al 2003 The spike gene was also optimized for codon us age in human cells to generate n spike The n spike gene was generated by PCR with overlapping oligonucleotides intro duced to optimize human codon usage The resulting overlap ping PCR fragments were fused and a full length codon opti mized spike cDNA was created Zhi et al 2005 The spike or n spike gene was cloned in pShuttle Clontech Palo Alto CA 3H11032 of the CMV promoter to generate pShCMV Spike and pShCMV nSpike respectively The CMV promoter was then replaced with the CAG promoter isolated from pCAGGS Niwa et al 1991 in both pShCMV Spike and pShCMV nSpike to generate pShCAG2 Spike and pShCAG2 nSpike respectively Pseudotyped HIV based vector was produced by triple trans fection of 293T cells using the CaPO 4 precipitation method as previously described Kobinger et al 2001 Briefly cells were transfected with the appropriate envelope expression vector the HIV packaging plasmid pCMVH9004R8 2 and the transfer vector pHxLacZWP and washed twice with serum free Dulbecco s modified Eagle s medium DMEM 16 hr later Pseudotyped virus like particles were concentrated from cell free supernatant by ultracentrifugation 60 hr posttransfection Vector was re suspended in complete DMEM and stored at H1100280 C Trans ducing units per milliliter TU ml was determined for each vec tor stock by counting H9252 Gal positive cells by limiting dilution on 293 T cells Vector stocks with titers of 1 6 H11003 10 8 TU ml for NTD6L HIV vector or 1 8 H11003 10 5 TU ml for spike HIV vec tor were used for experimentation All experiments involving the production and functional analysis of replication incompe tent pseudotyped HIV based vectors were performed under biosafety level 2H11001 containment as approved by the Wistar In stitute Philadelphia PA and the University of Pennsylvania Philadelphia PA Institutional Biosafety Committees Airway cultures Human airway cells were isolated from the trachea and main stem bronchi of donor lungs provided by the National Disease Research Exchange NDRI Philadelphia PA After enzy matic dispersion the cells were seeded on collagen coated semipermeable membrane supports Transwell COL 12 mm diameter and 0 4 H9262m pore size Corning Life Sciences Acton MA as previously described Kobinger et al 2001 Once cells reached confluence the apical medium was removed and the cells were maintained at an air liquid interface ALI to allow differentiation of the epithelial cell subpopulations Typ ically the ALI cultures were used 4 to 6 weeks after intro duction to air at which time transepithelial resistance was on the order of H11350500 H9024H11080 cm 2 To evaluate pseudotyped HIV based vectors ALI cultures were transduced with 100 H9262l of vector stock 1 6 H11003 10 7 LacZ 293T TU ml for NTD6L HIV vector or 4 8 H11003 10 4 293T TU ml for spike HIV vector ap plied to the apical side of the culture To exclude damage of epithelial integrity the transepithelial resistance was measured 24 hr after transduction at which time it remained at H11022500 H9024H11080cm 2 data not shown indicating that the epithelial integrity was not compromised The cultures were stained with 5 bromo 4 chloro 3 indolyl H9252 D galactopyranoside X Gal to reveal H9252 Gal expression 4 days after transduction H9252 Gal ex pressing cells were counted by examining 20 fields at H11003100 magnification and extrapolating for the surface area 1 cm 2 of the membrane support Bronchial explants Small portions 0 5 cm 2 of bronchi were excised from nor mal human airways and placed on collagen coated permeable supports Transwell COL 6 5 mm diameter and 0 4 H9262m pore size Corning Life Sciences The tissue was fed from the ba solateral reservoir Tissues were transduced with 50 100 H9262l of vector stock approximately 1 H11003 10 7 LacZ 293T TU ml for NTD6L HIV vector and 1 H11003 10 4 293T TU ml for spike HIV vector from the apical side and incubated for 2 4 hr at 37 C Fresh medium was then added so as to submerge the tissue Medium was replaced every 12 hr for 48 hr For H9252 Gal ex pression the tissue was fixed in 0 5 glutaraldehyde in phos phate buffered saline PBS for 10 min at room temperature and stained with X Gal at 37 C for 3 12 hr KOBINGER ET AL 414 Isolation and transduction of human monocyte derived dendritic cells Peripheral blood mononuclear cells isolated from healthy donors were obtained from the Center for AIDS Research CFAR facility at the University of Pennsylvania Monocytes adhered to a plastic surface by incubation for 2 hr at 37 C in AIM V medium Invitrogen Subsequently the nonadherent cells were removed and the plastic adherent monocytes were differentiated by application of a combination of granulocyte macrophage colony stimulating factor GM CSF 800 U ml Berlex Montville NJ and interleukin IL 4 500 U ml R boiled in a microwave for 6 min in 10 mM citrate buffer pH 6 0 treated sequentially with 2 H 2 O 2 avidin bi otin blocking reagents Vector Laboratories Burlingame CA and protein blocking agent Fisher Scientific Hampton NH followed by incubation with primary and biotinylated secondary antibodies Vector Laboratories Primary antibodies were di rected against the N terminus of the nucleocapsid N protein rabbit serum from Abgent San Diego CA diluted 1 50 Bound secondary antibodies were visualized with a VECTA STAIN Elite ABC kit Vector Laboratories and 3 3H11032 di aminobenzidine DAB as substrate In all cases incubation was followed by extensive washing with PBS Negative controls consisted of preincubation with PBS omission of the primary antibody and substitution of the primary antibody by an iso type matched nonimmune control antibody For electron mi croscopy cells were fixed in 2 5 glutaraldehyde in PBS and enclosed in 1 low melting point agarose After chilling on ice the cells in small agarose blocks were refixed overnight in 2 5 glutaraldehyde 2 paraformaldehyde in 0 1 M sodium ca codylate buffer pH 7 4 washed in cacodylate buffer and in cubated in 2 OsO 4 for 2 hr The samples were then stained for 1 hr with 1 uranyl acetate in 150 mM maleate buffer pH 6 0 washed dehydrated with ethanol and finally embedded in resin LX 112 Ladd Research Industries Williston VT Ul trathin sections 80 nm thick were stained with uranyl acetate and lead citrate according to standard protocols and samples were examined under a Philips CM 100 transmission electron microscope FEI Hillsboro OR Western blot 293T cells were transfected with each plasmid encoding spike protein using the CaPO 4 precipitation method described previously Forty eight hours later cells were harvested re suspended in lysis buffer and frozen at H1100220 C The total pro tein content of all samples determined by Bradford assay Bio Rad Hercules CA was normalized to the lysate with the lowest total protein by diluting with 4H11003 sodium dodecyl sul fate SDS sample buffer Invitrogen including 5 2 mercap toethanol Diluted samples were heated to 94 C for 4 min and 20 H9262g of each sample was loaded onto an SDS polyacrylamide gel After electrophoresis proteins were transferred onto a polyvinylidene difluoride PVDF membrane Blots were blocked with 8 milk diluted in PBS Tween 20 PBS T for 45 min washed three times 10 min each with PBS T and probed with primary antibody at room temperature for 1 hr Primary antibody consisted of whole serum isolated from rab bits inoculated with purified spike protein diluted in 5 milk PBS T to a final concentration of 1 500 The blots were then washed three times with PBS T before being incubated with secondary antibody for 45 min at room temperature and washed again three times with PBS T Secondary antibody was anti rabbit horseradish peroxidase HRP diluted in 5 milk PBS T to a final concentration of 1 2000 Santa Cruz Biotechnology Santa Cruz CA Protein bands were developed with SuperSignal West Pico chemiluminescent substrate Pierce Biotechnology Rockford IL and exposed on Kodak BioMax film Eastman Kodak Rochester NY RESULTS Generation of HIV based vector pseudotyped with the spike envelope protein of SARS CoV The gene encoding the spike envelope glycoprotein was gen erated by RT PCR from viral RNA isolated from strain Tor2 of SARS CoV The sequence of the spike gene was identical to the previously published sequence for the SARS CoV Tor2 isolate Marra et al 2003 The spike gene was cloned under the control of a CMV promoter in the pShuttle vector which TROPISM OF HIV VECTOR WITH SARS CoV ENVELOPE 415 was used to produce pseudotyped HIV based vector by triple transfection in 293T cells Initial yields of concentrated spike pseudotyped HIV based vector were on the order of 2 3 H11003 10 3 TU ml which is 5 6 logs lower than preparations of HIV based vector pseudotyped with VSV G or Zaire ebolavirus gly coprotein The production of spike HIV vector preparations with low titer was possibly due to weak intracellular expres sion of the spike glycoprotein or suboptimal packaging of the envelope into virus like particles Codon optimization was pre viously used to improve expression of spike and presumably vector titers from pseudotyped retroviral vector preparations Babcock et al 2004 Moore et al 2004 Yang et al 2004 Here intracellular expression of spike was enhanced both by codon optimizing the sequence for translation in mammalian cells n spike and by using the hybrid CAG promoter West ern blot analysis indicated that the expression of spike was sig nificantly increased by both codon optimization and the CAG promoter Fig 1A compare lanes 2 and 3 lanes 4 and 5 lanes 2 and 4 and lanes 3 and 5 respectively which resulted in a 2 to 3 log increase in the production of spike pseudotyped HIV transducing units per milliliter Fig 1B Subsequently pShCAG2 nSpike plasmid expressing the codon optimized spike under the control of the AG promoter was used to gen erate all working stocks of spike pseudotyped HIV based vec tor employed in this study A similar approach was used to op timize the expression of spike in adenovirus based vaccines Zhi et al 2005 Susceptibility of human airway epithelium to SARS CoV and spike pseudotyped HIV based vector Wild type SARS CoV was applied to the apical surface of human airway ALI cultures to address tropism of the virus for human conducting airway epithelia Infected ALI cultures were analyzed 10 days later for expression of SARS CoV antigen by immunohistochemical staining with a nucleocapsid N specific antibody Epithelial cells positive for N were detected in in fected cultures Fig 2B but not in cultures exposed only to ve hicle Fig 2A Analysis of SARS CoV infected ALI cultures by electron microscopy revealed the presence of progeny viri ons in the nucleus of epithelial cells Fig 2C and D Spike pseudotyped HIV based vector was evaluated next to assess whether relevant levels of transduction are possible with con centrated vector preparations Concentrated stock 100 H9262l per well containing 4 8 H11003 10 4 TU of spike pseudotyped HIV vec tor expressing the lacZ marker gene was added to the apical side of ALI cultures and H9252 Gal expression was analyzed 4 days later Controls included vector free DMEM and NTD6L pseudotyped HIV vector 1 6 H11003 10 7 TU well expressing LacZ previously shown to transduce ALI cultures Representative photomicrographs of cultures transduced with LacZ expressing vectors are shown in Fig 3 Spike pseudotyped HIV based vec tor demonstrated transduction of on average 70 75 up to 95 of the surface epithelium Fig 3C and F which was su perior to the 40 45 transduction efficiency obtained with NTD6L pseudotyped HIV vector Fig 3B and E no H9252 Gal ex pression was detected in ALI cultures exposed to vehicle Fig 3A and D To control for pseudo transduction experiments were carried out in the presence of zidovudine AZT 5 H9262M to inhibit RT and ablate vector encoded transgene Like uninfected cultures AZT treated cultures demonstrated no H9252 Gal expres sion indicating that the H9252 Gal activity is indeed the result of transduction rather than protein transfer data not shown These results indicate that primary cultures of human airway epithe lial cells are susceptible to SARS CoV and spike pseudotyped HIV vector KOBINGER ET AL 416 pShuttle Spike pShCAG2 Spike pShCAG2 nSpike T ransducing units ml 1 10 1 10 2 10 3 10 4 10 5 10 6 A B FIG 1 Optimization of spike expression and pseudotyped HIV based vector production A Expression of SARS CoV spike protein from three expression plasmids transfected into 293T cells See Materials and Methods for information regarding the plas mids Cell lysates were separated by SDS PAGE and expression of spike was detected by Western blotting using whole serum collected from rabbits inoculated with purified spike protein Mock transfected cell lysate served as a negative control Molecu lar weight markers are shown on the left The expected spike protein band is indicated arrow B Titers of LacZ expressing vector stock produced with various expression cassettes of spike Vector titers were determined by limiting dilution on 293T cells and are represented as transducing units per milliliter TU ml The column for each condition represents the mean of the four best titrating experiments among six to eight productions and each bar shows the standard deviation of the mean Transduction of human bronchial explants and murine lung airways by spike pseudotyped HIV based vector Excised sections of human bronchi were incubated for 2 4 hr with concentrated spike or NTD6L pseudotyped HIV vec tor encoding H9252 Gal to further characterize and compare the transduction efficiency of both vectors in intact airway epithe lium Tissue was fixed and stained with X Gal 16 hr after ap plication of the vector Spike pseudotyped vector resulted in ro bust expression of H9252 Gal in the surface epithelium stronger than with NTD6L pseudotyped vector Fig 4 compare panels C and F with panels B and E Histological photomicrographs of these tissues demonstrated no specific expression in the vehicle con trol a low level of expression in NTD6L pseudotyped vector treated tissue and strong expression of H9252 G