Human Molecular Genetics, 2001, Vol. 10, No. 26 3075-3081
© 2001 Oxford University Press
Exchange of surface proteins impacts on viral vector cellular specificity and transduction characteristics: the retina as a model
Institute for Human Gene Therapy, Department of Molecular and Cellular Engineering, The Wistar Institute and 1F.M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia, PA, USA
Received August 30, 2001; Revised and Accepted October 23, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIAL AND METHODSACKNOWLEDGMENTSREFERENCES |
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Recombinant vectors based on adeno-associated virus (AAV) or human immunodeficiency 1 (lentivirus) are promising tools for long term in vivo gene delivery. Their design allows the exchange of capsids or envelopes, respectively, theoretically providing the opportunity to transduce a range of cell types. We constructed AAV vectors encoding enhanced green fluorescent protein (EGFP) within an AAV serotype 2 (AAV2) genome contained in an AAV2, five or one capsid (called AAV2/2, AAV2/5 and AAV2/1, respectively). Similarly we produced lentiviral vectors, encoding the same expression cassette present in the AAV vectors, pseudotyped with proteins from vesicular stomatitis virus glycoprotein (VSVG) or Mokola envelopes. Transduction characteristics of these vectors were evaluated in the murine retina following subretinal or intravitreal administration. The time of onset of transgene expression and the targeted cell types differed between the various recombinants. Onset of transgene expression was 3–4 days for lentiviral vectors and AAV2/1. In contrast, onset was at 2–4 weeks for AAV2/5 and AAV2/2, respectively. After subretinal injection, both lenti-VSVG and AAV2/5 transduced the retinal pigment epithelium (RPE) and photoreceptors efficiently whereas transgene expression was restricted to RPE cells using lenti with the Mokola envelope or AAV2/1. After intravitreal administration, only AAV2/2 and lenti-VSVG transduced the inner retina. Vector-mediated fluorescence was detected in the retina for over 12 weeks for all of the vectors. We conclude that pseudotyping provides a useful means to manipulate viral vector cell targeting specificity as well as retinal transduction characteristics of vectors containing the same genome.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIAL AND METHODSACKNOWLEDGMENTSREFERENCES |
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Vectors based on adeno-associated virus (AAV) efficiently transduce various tissues in vivo and result in long-term expression (1). There are six primary isolates of AAV(types 1–6) (2–5). Recombinant AAV2 vectors, the best characterized, are currently used in a clinical trial for factor IX deficiency (6). The genomes of the different AAV isolates are organized similarly, with inverted terminal repeats (ITRs) flanking rep and cap-encoding regions. Their ITRs are similar so that an AAV2 genome can be packaged by any other serotype but 5 (4). Interestingly, AAV2 and five capsids share only 60% homology at the protein level and they bind to different receptors (4). This is reflected by the fact that cellular tropism differs between the two viruses, i.e. AAV5 transduces the airway epithelium from the apical side 50 times more efficiently than AAV2 (7). Also, in the CNS, the ability of AAV5 to infect post-mitotic neurons is increased compared to AAV2 (8). Similar comparisons in muscle indicate that vectors bearing an AAV1 capsid (2) result in higher levels of transduction compared to those derived from other serotypes (9). We recently described a strategy to package an AAV2 genome in an AAV5 capsid, based on a rep2–cap5 packaging construct (10). This allows direct comparisons of the effects of the capsids on transduction efficiency.
Retroviral vectors based on human immunodeficiency 1 (HIV-1) sequences, lentiviral vectors, are also able to transduce both dividing and non-dividing cells and they lead to sustained transgene expression from the integrated provirus (11). Lentiviruses are enveloped. Lentiviral vectors have been pseudotyped predominantly with the vesicular stomatitis virus glycoprotein (VSVG) which allows for production of high-titer recombinant virus able to enter a broad range of tissues, including the central nervous system and the retina (12–14). We recently demonstrated that exchanging VSVG with the envelope from the Ebola-Zaire strain of filovirus, allows for entry of the vector in murine airway epithelia cells in vivo from the apical side, which is resistant to VSVG-mediated entry (15). One possible explanation is that the apical side expresses a receptor specific for Ebola and not for VSVG.
The eye is an attractive organ for gene therapy applications for a number of reasons: it is enclosed and divided into small compartments, a situation which is ideal for precise delivery of vectors; it is an immune-privileged site, thus minimizing clearance of potentially immunogenic proteins; a number of inherited diseases affecting the eye have known gene defects; there are well characterized animal models for many of these conditions; and many of the diseases are slowly progressive, allowing for timely delivery of a potential treatment.
Several viral vectors target the retina (16). Among them, AAV type 2 (AAV2) and HIV-based vectors are particularly promising for retinal gene therapy, being able to lead to sustained, long-term expression of the transgene when delivered subretinally in various species (14,17,18). Initial efforts aimed at correcting mouse and more recently canine models of retinal degenerations using these vectors have been hopeful and invite further evaluation (19–23). Both AAV2 and VSVG-pseudotyped lentiviral vectors efficiently target retinal pigment epithelium (RPE) as well as photoreceptors following subretinal administration (14,20,21,23–27).
In an effort to enhance the potential for these viruses for gene therapy of both retinal degenerations and other blinding retinal diseases, we have designed strategies for targeting delivery of therapeutic genes to specific retinal cell types. We produced vectors containing an AAV2 genome in an AAV2, 5 or 1 capsid (AAV2/2, AAV2/5 and AAV2/1, respectively) as well as lentiviral vectors pseudotyped with proteins from VSVG and Mokola (28) envelopes. All vectors delivered the cDNA encoding the enhanced green fluorescent protein (EGFP) under the transcriptional control of the cytomegalovirus (CMV) promoter. These series of vectors allow us to analyze the effects of the capsid or envelope alone on transduction of the retina. We introduced the vectors independently in the murine retina and documented differences in onset, intensity and localization of transgene expression as well as the humoral response induced by the vectors.
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RESULTS |
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Onset and intensity of transgene expression following subretinal and intravitreal administration of the viral vectors
An aliquot (1 µl) of each high-infectious titer lentivirus [corresponding to 1.4 x 107 infectious units (IU) for VSVG and 6 x 105 IU for Mokola-pseudotyped vectors] or AAV2/2, AAV2/5, AAV2/1 and AAV5/5 large-scale preparations [corresponding to 1 x 109 genome copies (GC)] encoding EGFP under the control of the CMV promoter were injected either subretinally or intravitreally in C57Bl/6 mice. Ophthalmoscopic examination was performed 2, 4, 7, 14 and 28 days after vector administration and then once a month for 4 months (with the exception of AAV2/1 that has been followed for 4 weeks). EGFP expression was evident between 4 and 5 days after subretinal injection in the retinas administered with lentiviral vectors and AAV2/1, whereas the onset of transgene expression mediated by AAV2/5 or AAV5/5 is detected between day 14 and 21 (Table 1).
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For all vectors examined, expression was stable for over 90 days (and for 252 days for VSVG, the longest time points examined) following subretinal administration (Fig. 1). At the ophthalmoscopic level, strongest EGFP intensity was observed after subretinal delivery of AAV2/1–CMV–EGFP (Table 1).
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Following intravitreal administration of the vectors no fluorescence was detectable at the ophthalmoscopic level.
Localization of EGFP expression in the transduced retinas following subretinal administration
In order to identify which cell type was specifically transduced following subretinal administration of each viral vector, eyes were harvested at day 14 and 28, sectioned and analyzed under a fluorescent microscope.
Subretinal administration of AAV5/5– and AAV2/5–CMV–EGFP resulted in transduction of both RPE cells and photoreceptors (Fig. 2B and C) with higher levels of gene expression and percentage of transduced cells in the RPE layer. In comparison, AAV2/2 targets photoreceptors primarily and a smaller number of RPE cells (Fig. 2A). A similar pattern of transduction was observed upon administration of lenti-VSVG–CMV–EGFP (Fig. 2E). Delivery of lenti-Mokola– and AAV2/1–CMV–EGFP resulted in specific and efficient transduction of the RPE layer (Fig. 2D and F). No EGFP-positive photoreceptors were observed following transduction with lenti-Mokola. Some photoreceptors as well as Muller cells and cells in the inner nuclear layer (INL) were targeted by AAV2/1 but their fluorescence intensity was significantly lower than the RPE.
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Localization of EGFP expression in the transduced retinas following intravitreal administration
Histologic examination of the eyes administered intravitreally with the same set of vectors showed transduction of the lens epithelium (this may have been the result of injuring the lens capsule by the injection) and of the small blood vessels present in the inner half of the retina after delivery of lenti-VSVG (Fig. 3) and sparse EGFP-positive cells at the level of the ciliary body only in the retinas which received AAV5/5–, AAV2/5– and AAV2/1–CMV–EGFP (data not shown). Whereas expression in lens epithelium after injection of lenti-VSVG may have resulted from contacting the lens capsule during surgery, we did not see any evidence of that effect (i.e. there were neither cell infiltrate in the vitreous/anterior segment nor cataractous changes in the lens). Interestingly, the AAV2/2–CMV–EGFP vector, was able to efficiently transduce retinal ganglion cells (RGCs), Muller cells, the ciliary body and elements located in the INL, presumably amacrine cells (Fig. 4).
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Subretinal injections of all viruses induce a systemic humoral response to viral proteins and not to EGFP
Sera were obtained at the pre-treatment time point (day 0) and 14 days following subretinal injections of the vectors. Enzyme-linked immunoabsorbant assay (ELISA) analysis revealed that all viruses induced a systemic humoral response following both subretinal and intradermal administration (Table 2).
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Isotyping of the responses following subretinal administration of AAV- and HIV-based vectors revealed predominant induction of IgG2b and IgG1 levels for all viruses at 14 days following injection. On the contrary, intradermal administration of these viruses led to an overall increase in all the isotypes. However, the IgG2a levels were higher than IgG2b levels for each of the lentiviruses (Table 2).
Humoral responses (total IgG levels) against the transgenic (EGFP) were also evaluated following subretinal injections of the two lentiviruses and, in each case, was minimal (Table 3). Intradermal injections of the viruses induced only a mild antibody response against EGFP (data not shown).
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Characterization of the neutralizing antibody response to the viral vectors administered
For the neutralizing antibody (NAb) assay, 84-31 cells were incubated in the presence of virus and of serial dilutions of serum from animals injected either subretinally or intradermally with the same vector. The intensity of EGFP expression was captured digitally in the wells containing the virus-infected cells and was inversely proportional to the concentration of NAb. Therefore, an increase in EGFP expression, as assessed with fluorescence microscopy, correlates with a higher dilution of NAb titer detected with the Fluoroimager. Following subretinal administration (Table 4), the antibodies were non-neutralizing in nature.
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Interestingly, intradermal administration of our vectors results in production of NAbs (Table 4). This was reflected in the lower EGFP expression detected in the wells added with the same reciprocal dilutions of serum from animals administered with the vectors intradermally than subretinally (Table 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIAL AND METHODSACKNOWLEDGMENTSREFERENCES |
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In this study we demonstrate that the exchange of capsids between AAV serotypes or the pseudotyping of lentiviral vectors with proteins from other enveloped viruses results in transduction of distinct sets of cell types in the retina. These different vectors also impact on the onset and intensity of gene expression. Whereas AAV2/2 transduces photoreceptors efficiently, RPE is the preferential target of AAV2/5 and AAV5/5 (used as control) and the main target of AAV2/1. A similar difference in targeting specificity is observed following introduction of the vectors in the intravitreal space where AAV2/2 transduces the inner retina efficiently whereas AAV2/5 (and AAV5/5) or AAV2/1 result in poor expression in this region. The differences in cellular specificity using these vectors may reflect differences in the expression of viral receptors on the surface of various cell types. Whereas AAV2/2 is known to bind a receptor complex including heparan sulfate, integrins and fibroblast growth factor receptor 1 (29–31), AAV5/5 binding to the target cell requires 2,3-sialic acid (32). Little is known about AAV1/1 binding, but this appears to be different from the other serotypes tested.
Differences in transduction specificity are also observed after subretinal administration of differently pseudotyped HIV-based vectors. Robust EGFP expression is seen in both photoreceptors and RPE following subretinal injection of a lentiviral vector pseudotyped with VSVG. However, expression is restricted to the RPE when the same genome is packaged in the envelope protein from the Mokola virus. Although both are members of the rhabdoviridae family, VSVG and Mokola bind to different receptors, the phosphatidyl-serine and the acetylcholine receptor, respectively (33). Expression of the latter on RPE but not on photoreceptors may account for the pattern of transduction observed.
Exchanging capsids between AAV vectors impacts on targeting of the virus as well as on onset and intensity of gene expression. AAV2/2 and AAV2/5 lead to EGFP levels detectable by ophthalmoscopic examination in weeks. This delay in onset may reflect the time required for the single-strand genome to enter the nucleus and acquire a transcriptionally active status. Interestingly, the same genome in an AAV1 capsid leads to high levels of transgene expression detected ophthalmoscopically in only 4–5 days. This suggests that the capsid is a pivotal element in driving the biological intracellular events leading to efficient transduction.
The ability of vectors to transduce different cell types depending on their outer protein component has a direct implication for the treatment of diseases in which a specific cell is the site of the defect. Retinal degeneration, for instance, may develop from mutations in genes expressed specifically in photoreceptors or in the RPE (34). Previously, rhodopsin-specific promoters have been incorporated into AAV2/2 and lenti-VSVG in order to obtain expression restricted to photoreceptors (18,22,23). Vectors that selectively transduce a target cell can be used not only to provide the same specificity in terms of gene expression, but also to abrogate any safety concern arising from the presence of viral particles in cells that do not require gene transfer.
Upon intravitreal administration of AAV2/2, we observe efficient transduction not only of RGCs, as already described by Dudus et al. (27), but also of Muller cells, and amacrine cells (as suggested by their location in the INL). Cells in the ciliary body are also targeted to a lesser extent. VSVG instead targets the HIV-based vector to small vessels in the mid-retina. These are all appealing targets for gene transfer for diabetic proliferative retinopathy, the leading cause of blindness in adults between 21 and 74 years of age. In fact, progressive alterations of the retinal vessels lead to the hypoxia that triggers expression of vascular endothelial growth factor (VEGF) from RGC and Muller cells (35), thus resulting in inner retina neo-vascularization. AAV2/2 and lenti-VSVG may prove useful in bringing anti-angiogenic therapies to the specific sites where the pro-angiogenic stimulus is produced.
There was no evidence of toxic inflammatory response from clinical or histological examination using any of the vectors that was tested. One additional measure of toxicity is immune response. Subretinal administration of the pseudotyped lentiviral and AAV vectors resulted in the development of a systemic humoral response similar to that previously described for AAV2/2 (17,27,36). On isotyping, the antibodies primarily belonged to the Th2 isotype (IgG2b and IgG1). There was no evidence of a cell-mediated immune response. This finding is in agreement with our previous data (P.Karakousis et al., manuscript in preparation) supporting the hypothesis that the subretinal space can evoke an immune-deviant response similar to anterior chamber-associated immune deviation (37). The nature of the immune response to our viruses depends on the route of administration. Intradermal, a standard route for generating antivirus antibodies did indeed lead to production of NAbs. However, the subretinal injections were not associated with NAbs. The role of the unique environment of the eye with respect to antigen-specific immunity remains to be delineated.
In conclusion, we described and characterized novel agents for long-term gene delivery to the retina. In addition, we show that pseudotyping provides means with which to target viral vectors efficiently to specific retinal cell types and to influence levels and timing of gene expression. These findings, theoretically valid in other tissues, will have application to a variety of future studies, including evaluation of retinal development, generation of new animal models of ocular disease, and gene therapy for a diverse set of conditions including those resulting in retinal degeneration and retinal neovascularization.
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MATERIAL AND METHODS |
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Vector construction
The AAV5.EGFP expression plasmid pAAV5.CMV.EGFP3 was cloned as follows: the plasmid pAAV5 Rn lacZ (4) was subjected to PCR amplification using Pfu polymerase (Stratagene, La Jolla, CA) according to the manufacturer’s instructions using the primers 5'-TTCACAGCTTACAACATCTACAAAAC-3' (forward) and 5'-AAACTCGAGATTGCTAGCTCACTGCTTACAAAACCCCCTTGCTTGAG-3' (reverse), to provide plasmid pAAV5. pAAV5 was digested with the restriction enzymes NheI and XhoI, and the EGFP expression cassette in pAAV2.CMV.EGFP3 (38), isolated as a NheI–XhoI fragment, was inserted resulting in the plasmid pAAV5.CMV.EGFP3.
The AAV5 packaging construct pack5 was cloned as follows: viral AAV5 DNA was subjected to PCR amplification using Pfu polymerase (Stratagene) according to the manufacturer’s instructions using the primers 5'-cagtgatgtcataatgatgtaatgcttattgtcacgcga-3' (forward) and 5'-GATGTTGTAAGCTGTTATTCATTGAATGACCACAAGAGGCAGTATTTTA-3' (reverse), to provide the insert AAV5DITR. The insert was then subcloned into pZErO-2 (Invitrogen, Carlsbad, CA), providing plasmid pack5.
The AAV2/5 packaging construct including the AAV2rep and AAV5cap genes was already described by Hildinger et al. (10), as well as the AAV1 packaging construct (2) and the AAV2–CMV–EGFP (38).
Virus production
Recombinant AAV. Recombinant AAV2/2, AAV5/5, AAV2/5 and AAV2/1 viruses were produced as described elsewhere (10,38). Titers were determined by real-time PCR.
HIV-derived vectors. Pseudotyped HIV-based vector was generated by triple transfection of the helper packaging construct, the transfer vector and the envelope expressor as previously described by Kobinger et al. (15). Briefly, the helper packaging construct pCMVDR8.2 encoding the HIV helper function, the transfer vector pHR’EGFP encoding EGFP and plasmids encoding envelope proteins were used for triple transfection of 293T cells. Plasmids encoding the following viral envelopes were used to generate pseudotyped viruses: pMD.G (39) and pLTRMVG, encoding the Rhabdoviridae VSVG and Mokola (28) envelope proteins, respectively.
Triple transfection by using the CaPO4 precipitation method was performed as described by the manufacturer (Clontech, Palo Alto, CA) with 180 mg of endotoxin free DNA mixture applied to each 150 mm plate of 293T cells. Transfected cells were washed twice with serum-free DMEM 16 h later and cultured in DMEM 10% heat-inactivated FCS (complete DMEM) for an additional 15–20 h. Then, 11 ml of media was added to each 150 mm plate for harvesting of virus and media was allowed to remain on the plate for 16–20 h prior to collection. The media containing virus-like particles was filtered through a 0.45 µm filter and ultra-centrifuged at 28 000 r.p.m. in a Beckman SW28 rotor for 2 h at 4°C. Virus was resuspended in complete DMEM and stored at –80°C. Large-scale production from 20 150 mm plates transfected by CaPO4 yielded VSV-G-pseudotyped virus stock containing 5 x 109–1 x 1010 TU/ml upon concentration by ultra-centrifugation (as determined by EGFP expression using limiting dilution on 293T cells). All experiments involving the production and functional analysis of replication incompetent HIV-based pseudotyped vectors were performed under biosafety level 3 containment as approved by the Wistar Institute Institutional Biosafety Committee.
Animal studies
C57BL/6 mice (5–7 weeks old) (Taconic Farms, PA, USA) were used for all studies. Ten animals/vector were injected subretinally and the same number intravitreally. Additional sets of animals were injected intradermally. Animals were anesthetized and intraocular injections of each virus were performed as described previously by Bennett et al. (26). The same individual performed all the surgical procedures to minimize variability in injection technique. Four animals/vector/administration route were killed at day 14 and the same number at day 28, their eyes enucleated, fixed in 4% paraformaldheyde, transferred to 30% sucrose for 3–4 h and frozen in optimal cutting temperature compound (Fisher Scientific, Pittsburgh, PA). For each eye 150–200 10 µm serial sections were cut, the sections were progressively distributed on 10 slides so that each slide contains 15–20 sections representative of the whole eye at different levels. For each eye, three slides were analyzed. The images shown in the pictures are representative of the pattern and levels of expression seen histologically in the injected regions of eyes exposed to different vectors by different routes of administration. Under the conditions used, 20–30% of the retina is transduced following subretinal administration of each vector. Some sections were counterstained with propidium iodide or DAPI where indicated. All were mounted and analyzed under a fluorescence microscope.
EGFP-specific fluorescence was identified by co-visualization through FITC and rhodamine filters or through a triple filter where indicated. This allows simultaneous detection of EGFP (through the FITC filter) and background fluorescence (through the rhodamine and DAPI filters) (17,36). Indirect ophthalmoscopy was carried out as described elsewhere (26). The extent of retinal fluorescence was assessed with a numerical grading system ranging from 0 to 4. Grade 0 represents no visible fluorescence; grade 1, isolated fluorescing cells; grade 2, patches of fluorescing cells 1–2 disc areas in size; grade 3, confluent patches of fluorescing cells several disc areas in size, but requiring a condensing lens for visualization; grade 4, same as grade 3, but fluorescence is visible through the pupil without use of a condensing lens.
Enzyme-linked immunoabsorbant assay
Samples were analyzed for antibodies to viral capsid proteins and the transgene EGFP as previously described by Anand et al. (36). Briefly, enhanced protein binding ELISA plates (Corning, NY, USA) were coated overnight at 4°C with antigen using 109 GC/well of AAV5/5, AAV2/5 and AAV2/1 and 106 IU/well of lenti-VSVG– and lenti-Mokola–CMV–EGFP and 100 ng/well EGFP in bicarbonate buffer pH 9.6. Plates were then washed, blocked and incubated with diluted (1:100) serum. Saline and serum from an uninjected group of mice were used as negative control. The isotyping of the antibody response was performed by incubating with biotinylated anti-mouse IgG1, IgG2a, IgG2b and IgG3 (100 µl/well; Pharmingen, San Diego, CA and Becton Dickinson, Franklin Lakes, NJ) for 2 h. The wells were washed with phosphate-buffered saline containing 0.5% Tween and then incubated with 1:1000 diluted alkaline phosphatase-conjugated avidin (100 µl/well; Sigma, St Louis, MO) for 2 h. The wells were washed again and the color was developed using the Sigma Fast paranitrophenyl phosphate substrate (Sigma). The plates were read at an optical density of 405 nm. Measurements were repeated three times.
Identification of neutralizing antibodies
In order to identify NAbs, serum samples (10 µl) were plated in 96-well plates. Eight serial dilutions of each sample were made in serum-free DMEM. AAV5/5, AAV2/5 and AAV2/1 (2 x 108 IU), lenti-VSVG and lenti-Mokola viruses (2 x 106 IU) were added to each well, respectively, for NAb assays against each virus. The plates were incubated for 1 h at 37°C and then the samples were transferred to 96-well flat bottom tissue culture plates, which had been seeded with 84–31 cells (293 cells which express the E3 and E4 genes). The cells were 60% confluent. Infections were performed overnight. The following day, plates were inspected visually using a fluorescence microscope and analyzed using a Wallac Victor2 1420 Multilabel Counter (Perkin Elmer, Gaithersburg, MD). The NAb titer was defined as the highest dilution that allowed EGFP to be produced to levels 50% of control samples where no serum was added prior to virus infections.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIAL AND METHODSACKNOWLEDGMENTSREFERENCES |
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A.A. is a recipient of a fellowship from Telethon Italia (371/B). This study was supported by NIH RO1 EY10820 and EY12156 (J.B.), the Juvenile Diabetes Foundation, the Foundation Fighting Blindness, the Paul and Evanina Mackall Trust, the Lois Pope LIFE Foundation, the William and Mary Greve International Research Scholar Award (Research to Prevent Blindness, Inc.), the Ruth and Ann Milton Steinbach Fund and the F.M.Kirby Foundation.
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FOOTNOTES |
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+ To whom correspondence should be addressed at: Scheie Eye Institute, 310 Stellar-Chance Building, University of Pennsylvania, 422 Curie Boulevard, Philadelphia, PA 19104-6069, USA. Tel: +1 215 898 0915; Fax: +1 215 573 7155; Email: jebennet@mail.med.upenn.edu
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REFERENCES |
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