- Email: [email protected]
IIIa deleted adenovirus as a single-cycle genome replicating vector
Virology 462-463 (2014) 158–165
Contents lists available at ScienceDirect
Virology journal homepage: www.elsevier.com/locate/yviro
IIIa deleted adenovirus as a single-cycle genome replicating vector Catherine M. Crosby a, Michael A. Barry a,b,c,d,e,n a
Virology and Gene Therapy Graduate Program, Mayo Clinic, Rochester, MN 55902, USA Department of Internal Medicine, Division of Infectious Diseases, Mayo Clinic, Rochester, MN 55902, USA Translational Immunovirology and Biodefense Program, Mayo Clinic, Rochester, MN 55902, USA d Department of Immunology, Mayo Clinic, Rochester, MN 55902, USA e Department of Molecular Medicine, Mayo Clinic, Rochester, MN 55902, USA b c
art ic l e i nf o
a b s t r a c t
Article history: Received 9 April 2014 Returned to author for revisions 27 May 2014 Accepted 27 May 2014
Replication competent adenovirus (RC-Ad) vectors mediate robust transgene expression by virtue of amplifying transgenes by replication but also put patients at a risk of frank adenovirus infection. In contrast, E1-deleted replication defective Ad (RD-Ad) vectors are safer but produce substantially less transgene product. To generate a robust, but safer adenoviral vector, we created a “single cycle” adenovirus (SC-Ad) vector that replicates its genome and transgene, but that does not cause adenovirus infections by deleting the capsid cement protein IIIa in low seroprevalence adenovirus serotype 6. Ad6ΔIIIa can be produced in IIIa-expressing cell lines. In normal cells, Ad6-ΔIIIa replicates its genome and transgene but fails to package its DNA or form mature virus. SC-Ad and RC-Ad expressed transgenes hundreds of times higher than RD-Ad in human and mouse cells in vitro and in vivo in mice. These data suggest that SC-Ads may be safer amplifying vectors for vaccine and therapeutic applications. & 2014 Elsevier Inc. All rights reserved.
Keywords: Adenovirus Replication-competent Replication-defective Single-cycle Amplification Vaccines Gene therapy
Introduction Adenoviruses (Ads) are non-enveloped viruses carrying double-stranded DNA genomes (reviewed in (Campos and Barry, 2007)). Ad capsids are composed of seven proteins: hexon (II), penton base (III), fiber (IV), IIIa, VI, VIII and IX (Campos and Barry, 2007) (Fig. 1A). The major capsid proteins include hexon, penton base, and fiber that are present in 720, 60, and 36 copies per virion and make up the majority of the virion surface. Minor capsid proteins are IIIa, VI, VIII, and IX. Protein IIIa is a 63 kDa protein present at 60 copies per virion. IIIa was originally assigned to a position adjacent to the viral peripentonal hexons where it was thought to “cement” the interlocking facets together (Stewart et al., 1993) (Fig. 1A). While this may be its function, its position on the virion is still debated (Liu et al., 2010; Reddy et al., 2010). Ads were one of the first virus vector systems created for gene therapy or gene-based vaccine uses (Campos and Barry, 2007). A fully replication competent Ad (RC-Ad) gene-delivery vector poses significant safety concerns, due to the possibility of
n Corresponding author at: Mayo Clinic 200 First Street SW, Rochester, MN 55902, USA. Tel.: þ1 507 266 9090; fax: þ1 507 255 2811. E-mail address: [email protected] (M.A. Barry).
http://dx.doi.org/10.1016/j.virol.2014.05.030 0042-6822/& 2014 Elsevier Inc. All rights reserved.
uncontrolled viral replication and causing adenoviral disease. To circumvent this, replication defective vectors (RD-Ad) were developed by deleting pivotal E1 genes to drastically reduce viral genome replication, protein expression, and the production of progeny virions. Delivery of one RD-Ad to a cell delivers one copy of the transgene resulting in the expression of “1X” protein. In contrast, a replication-competent RC-Ad can replicate the same gene 100,000-fold to increase transgene expression and immune responses. When tested as HIV vaccines in macaques, RC-Ads are superior to RD-Ads, particularly by the oral route (Demberg et al., 2007; Demberg and Robert-Guroff, 2009; Gomez-Roman et al., 2006a; Gomez-Roman et al., 2006b; Gomez-Roman et al., 2006c; Hidajat et al., 2009; Malkevitch et al., 2003; Morgan et al., 2008; Patterson et al., 2004; Peng et al., 2006; Peng et al., 2005; Zhao et al., 2003; Zhao et al., 2005). While RC-Ads are more potent, they run the real risk of causing adenovirus infections. In contrast, a “single cycle” adenovirus that retains the ability to replicate its genome (and transgene), but is blocked from producing functional progeny virions may take advantage of transgene replication, but avoids the real risks of adenoviral infection. To test this concept, we deleted protein IIIa in species C adenovirus serotype 6 (Ad6) to create a single-cycle adenovirus (SC-Ad). In this work, we compare genome replication, virion production, infectivity, and the ability to express transgenes between RD-Ad6, SC-Ad6, and RC-Ad6.
C.M. Crosby, M.A. Barry / Virology 462-463 (2014) 158–165
159
Fig. 1. Adenovirus capsid and schematic of Ad genomes expressing GFP–Luc fusion protein. A) Diagram of adenovirus capsid. B) Schematic of Ad vectors used in the study. CMV-cytomegalovirus; ITR-Inverted terminal repeat.
Results Production of Ad6-ΔIIIa Protein IIIa is a minor cement protein thought to be located near the peripentonal hexons of the Ad capsid (Liu et al., 2010; Reddy et al., 2010) (Fig. 1A). The protein has roles in late stage virus maturation and serotype-specific genome packaging. Since these are steps that occur only at the later stages of capsid assembly after genome replication, we targeted IIIa as a potential viral modification to generate a SC-Ad. The majority of humans have pre-existing immunity to conventional adenovirus serotype 5 (Ad5) (Abbink et al., 2007). Ad6 is also a species of C adenovirus like Ad5 (Weaver et al., 2009; Weaver et al., 2013). However, Ad6 has lower seroprevalence than Ad5 and is not cross-neutralized by Ad5 antibodies. For these reasons, we deleted the gene for IIIA in Ad6 rather than Ad5. The IIIa gene was deleted in an E3-deleted Ad6 plasmid carrying a green fluorescence protein–luciferase fusion protein (GFPLuc) by homologous recombination in bacteria (Campos and Barry, 2004) (Fig. 1B). To provide IIIa in trans, the Ad6 IIIa cDNA was used to generate a 293 stable cell line (293-IIIa). IIIa expression was confirmed by western blot (data not shown). Ad6-ΔIIIa was rescued by transfection and propagation in 293-IIIa cells. Control Ad6 vectors with the same E3 deletion and GFPLuc transgene cassette were generated, including RD-Ad6 (with E1 deleted) and RC-Ad6 (with intact E1 and IIIa genes) (Fig. 1B). Ad6-ΔIIIa replicates its genome like RC-Ad6 To determine if Ad6-ΔIIIa was still able to replicate its genome, human A549 cells were infected with 1000 viral particles per cell (vp/cell) of it and RD-Ad and RC-Ad6. Two hours later, bound and excess virions were removed with trypsin and plated in 6 well dishes. To detect viral genomes, total DNA was isolated prior to
Fig. 2. Characterization of Ad6 vectors in A549 cells. A549 cells were infected with RD-, SC-, or RC-Ad6 at 1000 vp/cell. A) Total DNA was collected from cell lysates at 2 and 24 h post-infection. Viral DNA was quantified by qPCR with primers amplifying Ad6 hexon. B) MTT reagent was added to infected cells at the indicated time points. Developing reagent was added after 3.5 h and A595/620 was measured. Values represent % viability compared to mock. C) Total cell lysates were collected at the indicated time points. Samples were freeze-thawed 3 times and burst was quantified by TCID50 assay.
real time quantitative PCR against the hexon gene at 2 or 24 h (Fig. 2A). At 2 h, all three vectors had similar low levels of viral genomes associated with the cells. After 24 h, the viral genome copies for Ad6-ΔIIIa and RC-Ad6 increased 2000 to 3000-fold whereas RD-Ad6 copies did not amplify (Fig. 2A). Ad6-ΔIIIa kills infected cells but does not produce infectious progeny To determine if Ad6-ΔIIIa kills cells at a similar rate as RC-Ad6, cell viability was assessed by MTT assay. Cells infected with RDAd6 showed minimal loss of viability through day 4. Cells infected with Ad6-ΔIIIa or RC-Ad6 began to die by day 2, with increasing loss of viability through day 4 (Fig. 2B). There was no significant difference in viability between Ad6-ΔIIIa and RC-Ad6. While both viruses ultimately killed the cells, Ad6-ΔIIIa did not produce infectious progeny virions from A549 cells as assessed by viral burst assay (Fig. 2C).
Ad6-ΔIIIa produces immature, empty virus particles It was unclear whether Ad6-ΔIIIa could produce a virus particle in the absence of the IIIa protein. To test this, 293 and 293-IIIa
160
C.M. Crosby, M.A. Barry / Virology 462-463 (2014) 158–165
Fig. 3. SC-Ad6 particles of varied maturity formed in presence and absence of IIIa 293 (A, C) or 293-IIIa (B, D) were infected with SC-Ad6 at 500 vp/cell. After 2 days, cells were collected and divided. 5 106 cells were fixed and imaged by TEM. Images magnified 110,000x (A, B) and 220,000x (C, D).
cells were infected with Ad6-ΔIIIa at 500 vp/cell. 2 days later, the infected cells were analyzed by transmission electron microscopy (TEM) or were purified on CsCl gradients (Figs. 3 and 4). For TEM, a non-specific stain for electron dense regions was used. In addition to staining protein, cellular and viral DNA is darkly stained as well. TEM revealed a mixture of lightly and darkly stained particles in the nuclei of 293-IIIa cells (Fig. 3A and C). In the absence of IIIa in 293 cells, only lightly stained particles were observed (Fig. 3B and D). The major difference in the staining is in the internal core of the particle, where viral DNA would typically be packaged. When the Ad6-ΔIIIa infected 293-IIIa cells were lysed and separated on CsCl gradients, an upper “light” band and a lower “heavy” band corresponding to intermediate and mature particles, respectively (Fig. 4A). In contrast, lysates from Ad6-ΔIIIa infected 293 cells produced only a light CsCl band (Fig. 4B). Spectrophotometry at 260 and 280 nm and agarose gel electrophoresis indicated that the heavy band particles from 293-IIIa cells contained viral DNA, whereas the light intermediate particles from both cell lines did not contain viral genomic DNA (Fig. 4D).
Protein content of Ad6-ΔIIIa particles The light and heavy bands from the CsCl gradients were separated on 10% SDS-PAGE and the gel was stained with Coomassie blue to detect viral proteins (Fig. 4C). The heavy bands contained mature processed proteins VI and VII. In contrast, the light band particles from both 293-IIIa and 293 cells contained no mature proteins VI or VII. Instead, they contained only the precursor proteins pVI and pVIII indicating that the light band particles were immature intermediate adenoviral virions. As expected, Ad6-ΔIIIa light band particles produced from 293 cells contained no protein IIIa.
SC-Ad6 expresses more transgene product than RD-Ad6 in vitro These data indicated that Ad6-ΔIIIa behaved like a single-cycle adenovirus, so is hereafter referred to as SC-Ad6. The ultimate goal of the single cycle Ad vector is to express higher levels of transgene in cells compared to replication defective Ad vectors. The vectors in this study all encode GFP–Luc so expression could be easily monitored over time. Human A549 cells were infected at 1000 vp/cell, and luciferase expression was measured at the indicated time points (Fig. 5A). RD-Ad6 mediated low but increasing expression of luciferase. In contrast, cells infected with SC-Ad6 and RC-Ad6 produced 100-fold higher levels of luciferase than RD-Ad6 (p o0.0001 by repeated measures twoway ANOVA).
SC-Ad6 replicates and expresses more transgene product than RD-Ad6 in mice Mouse Hepa 1-6 cells are permissive for Ad replication and infection (Nagayama et al., 2003). Hepa 1-6 cells were infected at 1000 vp/cell and luciferase expression was monitored over time (Fig. 5B). In this case, luciferase expression increased over 2 days with SC-Ad and RC-Ad luciferase levels being 20 and 30 times higher than RD-Ad6 at this time point. In contrast to human A549 cells, luciferase expression in the mouse cells peaked at day 2 but then rapidly declined to RD-Ad6 levels by day 5. To characterize SC-Ad6 in vivo, groups of 5 BALB/c mice were injected intravenously with 3 1010 vp of the indicated viruses and livers were harvested on days one through four. qPCR of liver samples demonstrated that RD-Ad6 genome levels remained relatively constant over four days (Fig. 6A). In contrast, qPCR demonstrated that the genome copies of both SC-Ad6 and
C.M. Crosby, M.A. Barry / Virology 462-463 (2014) 158–165
161
Fig. 4. SC-Ad6 particles do not contain proteins VI, VII or IIIa. Infected 293-IIIa (A) and 293 (B) cells were separated on CsCl gradients. C) Purified virus particles were treated with SDS and run on a 10% SDS-PAGE, and stained with Bio-Safe Coomassie. D) Purified virus particles were treated with SDS and separated with agarose gel electrophoresis. A260/280 ratios for 293-empty: 1.04, 293-IIIa-empty: 1.11, 293-IIIa-mature: 1.26.
SC-Ad amplifies transgene expression in vivo in mice when compared to RD-Ad Liver samples that were collected for qPCR were also tested for luciferase activity (Fig. 6B). These data showed luciferase activity peaking at day 2 in the mice and declining through day 4. Consistent with genome amplification, SC-Ad and RC-Ad amplified transgene expressions above that mediated by the replicationdefective vector. In this case, SC-Ad6 and RC-Ad6 mediated 40and 300-fold higher luciferase activity than RD-Ad in the livers, respectively. SC-Ad and RC-Ad express more transgene product at lower multiplicities of infection than RD-Ad in primary human airway epithelia cells To determine how the vectors might behave in normal human cells, primary human small airway epithelial cells (HSAECs) were infected at varied multiplicities of infection with the GFPLuc expressing vectors and the cells were analyzed by microscopy 48 h later (Fig. 7). RD-Ad6 transduced most cells in the well at virus to cell ratios of 3.3 103 vp/cell. In contrast, both SC-Ad6 and RC-Ad6 generated at 33-fold lower multiplicities of infection. Fig. 5. Luciferase expression of Ad vectors in vitro. Human A549 (A) or mouse Hepa 1-6 (B) cells were infected at 1000 vp/cell. Luciferase expression was measured using Bright-Glo luciferase reagent at the indicated time points. Luminescence was measured using a Beckman Coulter plate reader.
RC-Ad6 were higher at all time points with an apparent peak at 2 days. At this peak, SC-Ad6 genome copies were 40-fold higher than RD-Ad6 while RC-Ad6 genomes were 125 times higher.
Discussion Human adenovirus vectors have shown potential for development as therapeutics for cancer, infectious disease, and genetic diseases. However, scale up into larger animals and humans and safety concerns have been significant hurdles for traditional replication defective and replication competent vectors. To overcome
162
C.M. Crosby, M.A. Barry / Virology 462-463 (2014) 158–165
Fig. 6. Genome amplification and luciferase expression in BALB/c mice. Groups of 30 female mice were injected with 3 1010 vp intravenously. At the indicated time points, liver samples were collected. (A) Total DNA was isolated and viral genomes were quantified by qPCR. N ¼ 15/virus group for day one, n¼ 5/virus group on days 2–4. (B) Samples were homogenized and Bright-Glo luciferase reagent was added at 1:1 ratio. Luminescence was measured using a Beckman Coulter plate reader. N ¼5/virus group/time point.
Fig. 7. GFP-mediated transduction of primary human airway epithelial cells. HSAECs were infected at the indicated vp/cell ratios and the monolayers were imaged on an inverted microscope 48 h later.
these challenges, we engineered “single cycle” replicating adenovirus 6 by deleting cement protein IIIa from the viral genome. SCAd6 effectively undergoes all of the early Ad life cycle events including early gene transcription and genome replication. The remaining late genes are transcribed and translated but in the absence of IIIa only empty, non-infectious virus particles are produced. This vector could have benefits over traditional replication defective and replication competent vectors. With RD-Ad6 infection, a low, but persistent level of luciferase protein is expressed both in vitro and in vivo. Due to the presence of E1 and other early genes, SC-Ad6 replicates its own genome along with any transgene it carries. In this proof of principle, SC-Ad6 produced significantly more luciferase than RD-Ad6 in vitro and in vivo in human and mouse cells. When the vectors were compared in human and mouse cells, the SC-Ad6 and RC-Ad6 were both able to replicate their genomes. This was interesting in part because it is generally accepted that adenoviruses do not replicate well in mice (Thomas et al., 2007). While it is true that infectious viruses are poorly replicated in mice, genome replication appears functional as demonstrated by qPCR. These data are consistent with early data showing that Ad5, another species C adenovirus, also amplifies in mouse liver after intravenous injection (Duncan et al., 1978). While the replicationcompetent Ad6 vectors did replicate their genomes and amplify transgene expression, these amplifications were approximately 50-fold less efficient than was observed in human cells. It should also be noted that luciferase expression in vitro and in vivo decreased after 2 days in mouse cells but not in human cells. It is unclear exactly why expression declined in mouse cells; however, this may be due to defects in the ability of human adenoviruses to complete the viral life cycle after genome replication. In particular, human Ads may fail to express late capsid components which may contribute to instability of newly synthesized viral genomes (Eggerding and Pierce, 1986). It is also possible that reduce late gene expression in mouse cells may perturb mouse cells and not humans cells to reduce expression. This is suggested in part by more rapid cell death in mouse Hepa1-6 cells than A549 cells even though SC- and RC-Ads replicate their genomes markedly higher in the human cells. From this, mice can be used to model single-cycle or replication-competent Ad6 but this model likely underestimates the impact of genome and transgene replication that may occur in non-human primates and humans. SC-Ad6 transgene expression was quite comparable with RCAd6 in human and mouse cell lines when tested in vitro. In contrast, in vivo, SC-Ad6 replication was 3-fold less efficient than RC-Ad6 and luciferase activity was 7-fold lower. These data suggest that factors present in vivo may modulate SC-Ad6 activity. These may be related to differences in genome packaging effects on innate immune responses or perhaps due to abortive second cycle infection by RC-Ad6 but not SC-Ad6. In contrast, when the vectors were tested in vitro on primary human airway cells, both SC-Ad and RC-Ad vectors generated markedly higher transduction at low multiplicities of infection. Notably, SC-Ad6 did not generate infectious virions making it appealing as safer replicating adenovirus vaccine than fully competent RC-Ads. While SC-Ad avoids a burst of infectious viruses from transduced cells, it does ultimately kill the initial target cells like RC-Ad. Therefore, these first infected cells are sacrificed to produce more transgene product. This makes SC-Ads less safe than RD-Ads but safer than RC-Ad6. This “Goldilocks” situation will require SC-Ads to be used under conditions that strike the right cost/benefit ratio. Sacrificing transduced cells is obviously not feasible for gene replacement therapy, so SC-Ads are not likely candidates for this type of therapy. In contrast, sacrificing mucosal cells for vaccines or tumor cells for cancer gene therapy may have merit.
C.M. Crosby, M.A. Barry / Virology 462-463 (2014) 158–165
In between these two extremes are using SC-Ads as gene-based vaccines, particularly for mucosal or oral vaccination. Fully wildtype RC-Ad4 and Ad7 adenoviruses have been used as oral vaccines to protect military personnel from respiratory infections. In this case, the vaccines must be delivered orally to avoid causing the respiratory disease one seeks to avoid (Couch et al., 1963). Oral RC-Ad delivery avoided the side effects of these wild viruses but still allowed mucosal vaccination to occur to protect from later RCAd respiratory infection. Therefore, RC-Ads applied to other sites run the real risk of causing adenovirus infections. In contrast, the SC-Ad vectors, can amplify antigen production during vaccination, but avoid the risk of adenovirus infection. They may particularly useful by the oral route, since most Ads are nearly quantitatively destroyed by stomach pH and digestive enzymes (Cheng et al., 2003). Therefore, RC-Ad4 and Ad7 vaccines were likely efficacious because they were able to amplify antigen production from the reduced number of cells that were infected by this stringent route. Proof of principle using RC-Ads safely in humans combined with the safer profile of SC-Ads, suggest these vectors may have utility also for systemic and mucosal vaccines against cancer and infectious agents.
Materials and methods Cell culture
163
amplified by PCR, adding BsaI sites on each end with compatible overhangs for MfeI. CMV-GFPLuc-SVA was inserted in pAd6PshSwa by direct ligation. pAd6-PshSwa-GL was linearized and recombined with pAd6-ΔE3. For RD-Ad generation, the E1 region was deleted by overlapping PCR of three fragments, one a 500bp region homologous to the region upstream of E1A, the second a blasticidin (BSD) resistance gene, and the third a 500 bp region homologous to pIX. The entire E1A/E1B region was removed by replacement with the BSD resistance gene by homologous recombination with pAd6-ΔE3-GL. For SC-Ad generation, the IIIa gene was deleted by overlapping PCR of three fragments, one a 500 bp region homologous to 52K, the second a zeocin resistance gene flanked by FRT sites, and the third a 500 bp region homologous to pII. The entire IIIa gene was replaced by the zeocin resistance gene, which was then removed with flp recombinase. E3 was deleted and GL was inserted by digesting pAd6-ΔE3-GL with PshAI and RsrII, and the E3 deletion and GL were recombined into pAd6ΔIIIa. Viruses were rescued in 293 cells or 293-IIIa cells and purified by double CsCl banding. Virus was desalted in 10% sucrose/KPBS. Virus particle concentration was determined by OD260. A260/280 ratios for each vector were: RD-Ad6-GL:1.3, SC-Ad6-GL:1.26, and RC-Ad6-GL:1.33. in vitro vector genome quantification 3 105 cells were plated in 6 well plates and infected at 1000 virus particles (vp)/cell. Two hours after infection, 1 ml of trypsin was added to cells. Detached cells were washed once with 5 ml of 5% FBS/DMEM. Cells were resuspended in 2 ml of 5% FBS/DMEM, and were plated in 6 well plates. After 24 h, cells were collected from plates with cell scrapers. Total DNA was isolated at 2–24 h after infection using the DNeasy Blood and Tissue kit according to manufacturer's protocol (Qiagen) with an RNase A digestion. Vector genomes were quantified using qPCR with primers against adenovirus hexon.
293 human embryonic kidney cells were purchased from Microbix (Toronto, Ontario, Canada). A549 lung carcinoma and mouse Hepa 1-6 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA). To produce 293-IIIa cells, the entire IIIa gene from Ad6 was PCR amplified and inserted into the plasmid pIRES-puro3 using BamHI and XhoI sites (Clontech, Mountain View, CA). The IIIa gene is expressed from a CMV promoter in a single transcript shared with an IRES-puromycin resistance gene. pIRES-puro3-IIIa was transfected in 293 cells using Polyfect reagent (Qiagen, Hilden, Germany), and cells were selected with 2 mg/ml puromycin. Cells were maintained as a mixed population, and IIIa expression was confirmed by western blot (data not shown). 293 cells were maintained in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS; HyClone, Rockford, IL) and penicillin/streptomycin at 100 U/mL (Gibco). Primary human small airway epithelial cells (HSAECs) were purchased from Lifeline Cell Technology (Frederick, MD). HSAECs were maintained in BronchiaLife SAE Complete Medium.
Concentration of DNA samples was determined by OD260 and diluted to 20 ng/μl. Real-time PCR was performed using the Applied Biosystems Prism 7900HT sequence detection system with SDS 2.3 software. Each well contained 10 μl Sybr Green (Applied Biosystems, Warrington, UK), 3.8 μl H2O, 0.6 μl of 10 μM F Primer, 0.6 μl of 10 μM R Primer, and 5 μl sample (i.e., 20ng DNA/ well).
Adenoviruses
Cell viability assay
A human adenovirus 6 plasmid was produced as described previously (Weaver et al., 2011), and genome modifications were completed using homologous recombination in bacteria as in Campos and Barry (2004). The E3 region of Ad6 was deleted by replacing the entire E3 region with a zeocin resistance gene. First, three PCR fragments were generated. The first was a 500 base pair product upstream of E3 and homologous to pVIII, the second a zeocin resistance gene flanked by short flippase recognition target (FRT) sites, and the third a 500 base pair product homologous downstream to gene U. A single DNA fragment was generated by overlapping PCR, which was recombined into pAd6. After recombination the zeocin gene was removed by flp recombinase in bacteria. A CMV-GFP–Luciferase-LZL-SV40 polyA cassette expressing a green fluorescence protein–luciferase fusion was inserted by first digesting pAd6 with PshAI and SwaI to make a smaller Ad6 plasmid with a unique MfeI site between the fiber and E4 open reading frames (pAd6-PshSwa). The CMV-GFPLuc-SVA gene was
Quantitative real time PCR (qPCR)
5 103 A549 cells were plated in 96 well plates and infected at 1000 vp/cell. At the indicated time points, 500 ml MTT (5 mg/ml) was added to the wells, and incubated 3.5 h at 37 1C. Media was removed and MTT developing reagent was added. After 15 min, A595 with A620 reference was read using the Beckman Coulter DTX 880 Multimode Detector system. in vitro virus burst assay 2 106 A549 cells were placed in a T75 flask and infected at 1000 vp/cell. 1 h post-infection, 3 mL trypsin was added to cells. Cells were washed once with 10 ml of 5% FBS/DMEM, and resuspended to a concentration of 2 105 cells/ml. 1 ml was collected and stored at 80 1C, and 1 ml was plated in each of 3 wells of a 6 well plate. Cells and supernatant were collected at indicated time points and stored at 80 1C. Cells were freezethawed 3 times and virus burst was quantified by TCID50 assay.
164
C.M. Crosby, M.A. Barry / Virology 462-463 (2014) 158–165
Transmission electron microscopy
Data Analysis
293 or 293-IIIa cells were infected with SC-Ad6 at 500 vp/cell. Cells were collected two days later and resuspended in 4% paraformaldehyde, 1% gluteraldehyde. Samples were processed and sectioned by the Electron Microscopy Core Facility (Mayo Clinic, Rochester, MN). Images were captured on a Philips CM10 Transmission Electron Microscope, equipped with a Gatan digital camera.
Graphs and statistical analyses were performed using Prism Graphical software.
SDS-PAGE and Coomassie blue staining 293 or 293-IIIa cells were infected with SC-Ad6 at 500 vp/cell. Cells were collected at two days, and virus was purified by single CsCl banding and desalted in 10% Sucrose/KPBS. Samples were boiled in 4x Laemelli's buffer, and run on a 10% tricine SDS-PAGE gel. The gel was stained with Bio-Safe Coomassie Stain (BioRad, Hercules, CA). Agarose gel electrophoresis of purified particles Equal numbers of purified virus particles were combined with 0.2% SDS at a Virus:PBS:SDS ratio of 1:1:2. Samples were combined with 6x DNA loading dye, and separated on a 0.8% agarose gel containing GelRed nucleic acid stain (Biotium, Hayward, CA). in vitro luciferase assay To quantify luciferase expression 1 103 cells were plated in black-walled 96-well plates and infected at 1000 vp/cell. At indicated time points, Bright-Glo luciferase reagent (Promega, Madison, WI) was added at a 1:1 ratio and luciferase activity was measured using the Beckman Coulter DTX 880 Multimode Detector system. Animals Female BALB/c mice were purchased from Harlan SpragueDawley (Indianapolis, IN). They were housed in the Mayo Clinic Animal Facility under the Association for Assessment and Accreditation of Laboratory Animal Care (AALAC) guidelines with animal use protocols approved by the Mayo Clinic Animal Use and Care Committee. All animal experiments were carried out according to the provisions of the Animal Welfare Act, PHS Animal Welfare Policy, the principles of the NIH Guide for the Care and Use of Laboratory Animals, and the policies and procedures of Mayo Clinic. Mice were injected intravenously with 3 1010 vp in 100 μL PBS. DNA isolation from mouse liver tissues Liver samples were collected and washed with PBS. Total DNA was isolated from liver samples using a Maxwell 16 DNA Purification Kit (Promega, Madison, WI). Luciferase expression of mouse liver tissues Liver samples were collected and washed with PBS. Samples were homogenized in T-Per Reagent containing protease inhibitors (Fisher Scientific, Rockford, IL). Homogenized tissue and Bright-Glo luciferase reagent (Promega, Madison, WI) were added at a 1:1 ratio of volume. Luciferase activity was measured using the Beckman Coulter DTX 880 Multimode Detector system.
Acknowledgments We would like to thank Mary Barry for excellent technical assistance and Tien Nguyen with assistance in processing liver samples. We also would like to thank Samuel Campos for originally generating the IIIa deletion construct. This work was supported by the Electron Microscopy Core Facility at Mayo Clinic. This work was supported by NIH/NIAID Grants R01 AI096967 and R01 CA136945.
References Abbink, P., Lemckert, A.A., Ewald, B.A., Lynch, D.M., Denholtz, M., Smits, S., Holterman, L., Damen, I., Vogels, R., Thorner, A.R., O'Brien, K.L., Carville, A., Mansfield, K.G., Goudsmit, J., Havenga, M.J., Barouch, D.H., 2007. Comparative seroprevalence and immunogenicity of six rare serotype recombinant adenovirus vaccine vectors from subgroups B and D. J. Virol. 81, 4654–4663. Campos, S.K., Barry, M.A., 2004. Rapid construction of capsid-modified adenoviral vectors through bacteriophage lambda red recombination. Hum. Gene Ther. 15, 1125–1130. Campos, S.K., Barry, M.A., 2007. Current advances and future challenges in Adenoviral vector biology and targeting. Curr. Gene Ther. 7, 189–204. Cheng, X., Ming, X., Croyle, M.A., 2003. PEGylated adenoviruses for gene delivery to the intestinal epithelium by the oral route. Pharm. Res. 20, 1444–1451. Couch, R.B., Chanock, R.M., Cate, T.R., Lang, D.J., Knight, V., Huebner, R.J., 1963. Immunization with types 4 and 7 adenovirus by selective infection of the intestinal tract. Am. Rev. Respir. Dis. 88 (Suppl.), 394–403. Demberg, T., Florese, R.H., Heath, M.J., Larsen, K., Kalisz, I., Kalyanaraman, V.S., Lee, E.M., Pal, R., Venzon, D., Grant, R., Patterson, L.J., Korioth-Schmitz, B., Buzby, A., Dombagoda, D., Montefiori, D.C., Letvin, N.L., Cafaro, A., Ensoli, B., RobertGuroff, M., 2007. A replication-competent adenovirus-human immunodeficiency virus (Ad-HIV) tat and Ad-HIV env priming/Tat and envelope protein boosting regimen elicits enhanced protective efficacy against simian/human immunodeficiency virus SHIV89.6P challenge in rhesus macaques. J. Virol. 81, 3414–3427. Demberg, T., Robert-Guroff, M., 2009. Mucosal immunity and protection against HIV/SIV infection: strategies and challenges for vaccine design. Int. Rev. Immunol. 28, 20–48. Duncan, S.J., Gordon, F.C., Gregory, D.W., McPhie, J.L., Postlethwaite, R., White, R., Willcox, H.N., 1978. Infection of mouse liver by human adenovirus type 5. J. Gen. Virol. 40, 45–61. Eggerding, F.A., Pierce, W.C., 1986. Molecular biology of adenovirus type 2 semipermissive infections. I. Viral growth and expression of viral replicative functions during restricted adenovirus infection. Virology 148, 97–113. Gomez-Roman, V.R., Florese, R.H., Patterson, L.J., Peng, B., Venzon, D., Aldrich, K., Robert-Guroff, M., 2006a. A simplified method for the rapid fluorometric assessment of antibody-dependent cell-mediated cytotoxicity. J. Immunol. Methods 308, 53–67. Gomez-Roman, V.R., Florese, R.H., Peng, B., Montefiori, D.C., Kalyanaraman, V.S., Venzon, D., Srivastava, I., Barnett, S.W., Robert-Guroff, M., 2006b. An adenovirus-based HIV subtype B prime/boost vaccine regimen elicits antibodies mediating broad antibody-dependent cellular cytotoxicity against nonsubtype B HIV strains. J. Acquir. Immune Defic. Syndr. 43, 270–277. Gomez-Roman, V.R., Grimes Jr., G.J., Potti, G.K., Peng, B., Demberg, T., Gravlin, L., Treece, J., Pal, R., Lee, E.M., Alvord, W.G., Markham, P.D., Robert-Guroff, M., 2006c. Oral delivery of replication-competent adenovirus vectors is well tolerated by SIV- and SHIV-infected rhesus macaques. Vaccine 24, 5064–5072. Hidajat, R., Xiao, P., Zhou, Q., Venzon, D., Summers, L.E., Kalyanaraman, V.S., Montefiori, D.C., Robert-Guroff, M., 2009. Correlation of vaccine-elicited systemic and mucosal non-neutralizing antibody activities with reduced acute viremia following intrarectal SIVmac251 challenge of rhesus macaques. J. Virol. 83 (2), 791–801. Liu, H., Jin, L., Koh, S.B., Atanasov, I., Schein, S., Wu, L., Zhou, Z.H., 2010. Atomic structure of human adenovirus by cryo-EM reveals interactions among protein networks. Science 329 (5995), 1038–1043. Malkevitch, N., Patterson, L.J., Aldrich, K., Richardson, E., Alvord, W.G., RobertGuroff, M., 2003. A replication competent adenovirus 5 host range mutantsimian immunodeficiency virus (SIV) recombinant priming/subunit protein boosting vaccine regimen induces broad, persistent SIV-specific cellular immunity to dominant and subdominant epitopes in Mamu-A*01 rhesus macaques. J. Immunol. 170, 4281–4289. Morgan, C., Marthas, M., Miller, C., Duerr, A., Cheng-Mayer, C., Desrosiers, R., Flores, J., Haigwood, N., Hu, S.L., Johnson, R.P., Lifson, J., Montefiori, D., Moore, J.,
C.M. Crosby, M.A. Barry / Virology 462-463 (2014) 158–165
Robert-Guroff, M., Robinson, H., Self, S., Corey, L., 2008. The use of nonhuman primate models in HIV vaccine development. PLoS Med. 5, e173. Nagayama, Y., Nakao, K., Mizuguchi, H., Hayakawa, T., Niwa, M., 2003. Enhanced antitumor effect of combined replicative adenovirus and nonreplicative adenovirus expressing interleukin-12 in an immunocompetent mouse model. Gene Ther. 10, 1400–1403. Patterson, L.J., Malkevitch, N., Venzon, D., Pinczewski, J., Gomez-Roman, V.R., Wang, L., Kalyanaraman, V.S., Markham, P.D., Robey, F.A., Robert-Guroff, M., 2004. Protection against mucosal simian immunodeficiency virus SIV(mac251) challenge by using replicating adenovirus-SIV multigene vaccine priming and subunit boosting. J. Virol. 78, 2212–2221. Peng, B., Voltan, R., Cristillo, A.D., Alvord, W.G., Davis-Warren, A., Zhou, Q., Murthy, K.K., Robert-Guroff, M., 2006. Replicating Ad-recombinants encoding nonmyristoylated rather than wild-type HIV Nef elicit enhanced cellular immunity. AIDS 20, 2149–2157. Peng, B., Wang, L.R., Gomez-Roman, V.R., Davis-Warren, A., Montefiori, D.C., Kalyanaraman, V.S., Venzon, D., Zhao, J., Kan, E., Rowell, T.J., Murthy, K.K., Srivastava, I., Barnett, S.W., Robert-Guroff, M., 2005. Replicating rather than nonreplicating adenovirus-human immunodeficiency virus recombinant vaccines are better at eliciting potent cellular immunity and priming high-titer antibodies. J. Virol. 79, 10200–10209. Reddy, V.S., Natchiar, S.K., Stewart, P.L., Nemerow, G.R., 2010. Crystal structure of human adenovirus at 3.5 A resolution. Science 329 (5995), 1071–1075. Stewart, P.L., Fuller, S.D., Burnett, R.M., 1993. Difference imaging of adenovirus: bridging the resolution gap between X-ray crystallography and electron microscopy. EMBO J. 12, 2589–2599.
165
Thomas, M.A., Spencer, J.F., Wold, W.S., 2007. Use of the Syrian hamster as an animal model for oncolytic adenovirus vectors. Methods Mol. Med. 130, 169–183. Weaver, E.A., Nehete, P.N., Buchl, S.S., Senac, J.S., Palmer, D., Ng, P., Sastry, K.J., Barry, M.A., 2009. Comparison of replication-competent, first generation, and helperdependent adenoviral vaccines. PloS One 4, e5059. Weaver, E.A., Nehete, P.N., Nehete, B.P., Yang, G., Buchl, S.J., Hanley, P.W., Palmer, D., Montefiori, D.C., Ferrari, G., Ng, P., Sastry, K.J., Barry, M.A., 2013. Comparison of systemic and mucosal immunization with helper-dependent adenoviruses for vaccination against mucosal challenge with SHIV. PloS One 8 (7), e67574, http://dx.doi.org/10.1371/journal.pone.0067574. Weaver, E.A., Palmer, D., Ng, P., Barry, M.A., 2011. Characterization of species C human adenovirus serotype 6 (Ad6). Virology 412 (1), 19–27, http://dx.doi.org/ 10.1016/j.virol.2010.10.041. Zhao, J., Lou, Y., Pinczewski, J., Malkevitch, N., Aldrich, K., Kalyanaraman, V.S., Venzon, D., Peng, B., Patterson, L.J., Edghill-Smith, Y., Woodward, R., Pavlakis, G. N., Robert-Guroff, M., 2003. Boosting of SIV-specific immune responses in rhesus macaques by repeated administration of Ad5hr-SIVenv/rev and Ad5hrSIVgag recombinants. Vaccine 21, 4022–4035. Zhao, J., Voltan, R., Peng, B., Davis-Warren, A., Kalyanaraman, V.S., Alvord, W.G., Aldrich, K., Bernasconi, D., Butto, S., Cafaro, A., Ensoli, B., Robert-Guroff, M., 2005. Enhanced cellular immunity to SIV Gag following co-administration of adenoviruses encoding wild-type or mutant HIV Tat and SIV Gag. Virology 342, 1–12.
Contents lists available at ScienceDirect
Virology journal homepage: www.elsevier.com/locate/yviro
IIIa deleted adenovirus as a single-cycle genome replicating vector Catherine M. Crosby a, Michael A. Barry a,b,c,d,e,n a
Virology and Gene Therapy Graduate Program, Mayo Clinic, Rochester, MN 55902, USA Department of Internal Medicine, Division of Infectious Diseases, Mayo Clinic, Rochester, MN 55902, USA Translational Immunovirology and Biodefense Program, Mayo Clinic, Rochester, MN 55902, USA d Department of Immunology, Mayo Clinic, Rochester, MN 55902, USA e Department of Molecular Medicine, Mayo Clinic, Rochester, MN 55902, USA b c
art ic l e i nf o
a b s t r a c t
Article history: Received 9 April 2014 Returned to author for revisions 27 May 2014 Accepted 27 May 2014
Replication competent adenovirus (RC-Ad) vectors mediate robust transgene expression by virtue of amplifying transgenes by replication but also put patients at a risk of frank adenovirus infection. In contrast, E1-deleted replication defective Ad (RD-Ad) vectors are safer but produce substantially less transgene product. To generate a robust, but safer adenoviral vector, we created a “single cycle” adenovirus (SC-Ad) vector that replicates its genome and transgene, but that does not cause adenovirus infections by deleting the capsid cement protein IIIa in low seroprevalence adenovirus serotype 6. Ad6ΔIIIa can be produced in IIIa-expressing cell lines. In normal cells, Ad6-ΔIIIa replicates its genome and transgene but fails to package its DNA or form mature virus. SC-Ad and RC-Ad expressed transgenes hundreds of times higher than RD-Ad in human and mouse cells in vitro and in vivo in mice. These data suggest that SC-Ads may be safer amplifying vectors for vaccine and therapeutic applications. & 2014 Elsevier Inc. All rights reserved.
Keywords: Adenovirus Replication-competent Replication-defective Single-cycle Amplification Vaccines Gene therapy
Introduction Adenoviruses (Ads) are non-enveloped viruses carrying double-stranded DNA genomes (reviewed in (Campos and Barry, 2007)). Ad capsids are composed of seven proteins: hexon (II), penton base (III), fiber (IV), IIIa, VI, VIII and IX (Campos and Barry, 2007) (Fig. 1A). The major capsid proteins include hexon, penton base, and fiber that are present in 720, 60, and 36 copies per virion and make up the majority of the virion surface. Minor capsid proteins are IIIa, VI, VIII, and IX. Protein IIIa is a 63 kDa protein present at 60 copies per virion. IIIa was originally assigned to a position adjacent to the viral peripentonal hexons where it was thought to “cement” the interlocking facets together (Stewart et al., 1993) (Fig. 1A). While this may be its function, its position on the virion is still debated (Liu et al., 2010; Reddy et al., 2010). Ads were one of the first virus vector systems created for gene therapy or gene-based vaccine uses (Campos and Barry, 2007). A fully replication competent Ad (RC-Ad) gene-delivery vector poses significant safety concerns, due to the possibility of
n Corresponding author at: Mayo Clinic 200 First Street SW, Rochester, MN 55902, USA. Tel.: þ1 507 266 9090; fax: þ1 507 255 2811. E-mail address: [email protected] (M.A. Barry).
http://dx.doi.org/10.1016/j.virol.2014.05.030 0042-6822/& 2014 Elsevier Inc. All rights reserved.
uncontrolled viral replication and causing adenoviral disease. To circumvent this, replication defective vectors (RD-Ad) were developed by deleting pivotal E1 genes to drastically reduce viral genome replication, protein expression, and the production of progeny virions. Delivery of one RD-Ad to a cell delivers one copy of the transgene resulting in the expression of “1X” protein. In contrast, a replication-competent RC-Ad can replicate the same gene 100,000-fold to increase transgene expression and immune responses. When tested as HIV vaccines in macaques, RC-Ads are superior to RD-Ads, particularly by the oral route (Demberg et al., 2007; Demberg and Robert-Guroff, 2009; Gomez-Roman et al., 2006a; Gomez-Roman et al., 2006b; Gomez-Roman et al., 2006c; Hidajat et al., 2009; Malkevitch et al., 2003; Morgan et al., 2008; Patterson et al., 2004; Peng et al., 2006; Peng et al., 2005; Zhao et al., 2003; Zhao et al., 2005). While RC-Ads are more potent, they run the real risk of causing adenovirus infections. In contrast, a “single cycle” adenovirus that retains the ability to replicate its genome (and transgene), but is blocked from producing functional progeny virions may take advantage of transgene replication, but avoids the real risks of adenoviral infection. To test this concept, we deleted protein IIIa in species C adenovirus serotype 6 (Ad6) to create a single-cycle adenovirus (SC-Ad). In this work, we compare genome replication, virion production, infectivity, and the ability to express transgenes between RD-Ad6, SC-Ad6, and RC-Ad6.
C.M. Crosby, M.A. Barry / Virology 462-463 (2014) 158–165
159
Fig. 1. Adenovirus capsid and schematic of Ad genomes expressing GFP–Luc fusion protein. A) Diagram of adenovirus capsid. B) Schematic of Ad vectors used in the study. CMV-cytomegalovirus; ITR-Inverted terminal repeat.
Results Production of Ad6-ΔIIIa Protein IIIa is a minor cement protein thought to be located near the peripentonal hexons of the Ad capsid (Liu et al., 2010; Reddy et al., 2010) (Fig. 1A). The protein has roles in late stage virus maturation and serotype-specific genome packaging. Since these are steps that occur only at the later stages of capsid assembly after genome replication, we targeted IIIa as a potential viral modification to generate a SC-Ad. The majority of humans have pre-existing immunity to conventional adenovirus serotype 5 (Ad5) (Abbink et al., 2007). Ad6 is also a species of C adenovirus like Ad5 (Weaver et al., 2009; Weaver et al., 2013). However, Ad6 has lower seroprevalence than Ad5 and is not cross-neutralized by Ad5 antibodies. For these reasons, we deleted the gene for IIIA in Ad6 rather than Ad5. The IIIa gene was deleted in an E3-deleted Ad6 plasmid carrying a green fluorescence protein–luciferase fusion protein (GFPLuc) by homologous recombination in bacteria (Campos and Barry, 2004) (Fig. 1B). To provide IIIa in trans, the Ad6 IIIa cDNA was used to generate a 293 stable cell line (293-IIIa). IIIa expression was confirmed by western blot (data not shown). Ad6-ΔIIIa was rescued by transfection and propagation in 293-IIIa cells. Control Ad6 vectors with the same E3 deletion and GFPLuc transgene cassette were generated, including RD-Ad6 (with E1 deleted) and RC-Ad6 (with intact E1 and IIIa genes) (Fig. 1B). Ad6-ΔIIIa replicates its genome like RC-Ad6 To determine if Ad6-ΔIIIa was still able to replicate its genome, human A549 cells were infected with 1000 viral particles per cell (vp/cell) of it and RD-Ad and RC-Ad6. Two hours later, bound and excess virions were removed with trypsin and plated in 6 well dishes. To detect viral genomes, total DNA was isolated prior to
Fig. 2. Characterization of Ad6 vectors in A549 cells. A549 cells were infected with RD-, SC-, or RC-Ad6 at 1000 vp/cell. A) Total DNA was collected from cell lysates at 2 and 24 h post-infection. Viral DNA was quantified by qPCR with primers amplifying Ad6 hexon. B) MTT reagent was added to infected cells at the indicated time points. Developing reagent was added after 3.5 h and A595/620 was measured. Values represent % viability compared to mock. C) Total cell lysates were collected at the indicated time points. Samples were freeze-thawed 3 times and burst was quantified by TCID50 assay.
real time quantitative PCR against the hexon gene at 2 or 24 h (Fig. 2A). At 2 h, all three vectors had similar low levels of viral genomes associated with the cells. After 24 h, the viral genome copies for Ad6-ΔIIIa and RC-Ad6 increased 2000 to 3000-fold whereas RD-Ad6 copies did not amplify (Fig. 2A). Ad6-ΔIIIa kills infected cells but does not produce infectious progeny To determine if Ad6-ΔIIIa kills cells at a similar rate as RC-Ad6, cell viability was assessed by MTT assay. Cells infected with RDAd6 showed minimal loss of viability through day 4. Cells infected with Ad6-ΔIIIa or RC-Ad6 began to die by day 2, with increasing loss of viability through day 4 (Fig. 2B). There was no significant difference in viability between Ad6-ΔIIIa and RC-Ad6. While both viruses ultimately killed the cells, Ad6-ΔIIIa did not produce infectious progeny virions from A549 cells as assessed by viral burst assay (Fig. 2C).
Ad6-ΔIIIa produces immature, empty virus particles It was unclear whether Ad6-ΔIIIa could produce a virus particle in the absence of the IIIa protein. To test this, 293 and 293-IIIa
160
C.M. Crosby, M.A. Barry / Virology 462-463 (2014) 158–165
Fig. 3. SC-Ad6 particles of varied maturity formed in presence and absence of IIIa 293 (A, C) or 293-IIIa (B, D) were infected with SC-Ad6 at 500 vp/cell. After 2 days, cells were collected and divided. 5 106 cells were fixed and imaged by TEM. Images magnified 110,000x (A, B) and 220,000x (C, D).
cells were infected with Ad6-ΔIIIa at 500 vp/cell. 2 days later, the infected cells were analyzed by transmission electron microscopy (TEM) or were purified on CsCl gradients (Figs. 3 and 4). For TEM, a non-specific stain for electron dense regions was used. In addition to staining protein, cellular and viral DNA is darkly stained as well. TEM revealed a mixture of lightly and darkly stained particles in the nuclei of 293-IIIa cells (Fig. 3A and C). In the absence of IIIa in 293 cells, only lightly stained particles were observed (Fig. 3B and D). The major difference in the staining is in the internal core of the particle, where viral DNA would typically be packaged. When the Ad6-ΔIIIa infected 293-IIIa cells were lysed and separated on CsCl gradients, an upper “light” band and a lower “heavy” band corresponding to intermediate and mature particles, respectively (Fig. 4A). In contrast, lysates from Ad6-ΔIIIa infected 293 cells produced only a light CsCl band (Fig. 4B). Spectrophotometry at 260 and 280 nm and agarose gel electrophoresis indicated that the heavy band particles from 293-IIIa cells contained viral DNA, whereas the light intermediate particles from both cell lines did not contain viral genomic DNA (Fig. 4D).
Protein content of Ad6-ΔIIIa particles The light and heavy bands from the CsCl gradients were separated on 10% SDS-PAGE and the gel was stained with Coomassie blue to detect viral proteins (Fig. 4C). The heavy bands contained mature processed proteins VI and VII. In contrast, the light band particles from both 293-IIIa and 293 cells contained no mature proteins VI or VII. Instead, they contained only the precursor proteins pVI and pVIII indicating that the light band particles were immature intermediate adenoviral virions. As expected, Ad6-ΔIIIa light band particles produced from 293 cells contained no protein IIIa.
SC-Ad6 expresses more transgene product than RD-Ad6 in vitro These data indicated that Ad6-ΔIIIa behaved like a single-cycle adenovirus, so is hereafter referred to as SC-Ad6. The ultimate goal of the single cycle Ad vector is to express higher levels of transgene in cells compared to replication defective Ad vectors. The vectors in this study all encode GFP–Luc so expression could be easily monitored over time. Human A549 cells were infected at 1000 vp/cell, and luciferase expression was measured at the indicated time points (Fig. 5A). RD-Ad6 mediated low but increasing expression of luciferase. In contrast, cells infected with SC-Ad6 and RC-Ad6 produced 100-fold higher levels of luciferase than RD-Ad6 (p o0.0001 by repeated measures twoway ANOVA).
SC-Ad6 replicates and expresses more transgene product than RD-Ad6 in mice Mouse Hepa 1-6 cells are permissive for Ad replication and infection (Nagayama et al., 2003). Hepa 1-6 cells were infected at 1000 vp/cell and luciferase expression was monitored over time (Fig. 5B). In this case, luciferase expression increased over 2 days with SC-Ad and RC-Ad luciferase levels being 20 and 30 times higher than RD-Ad6 at this time point. In contrast to human A549 cells, luciferase expression in the mouse cells peaked at day 2 but then rapidly declined to RD-Ad6 levels by day 5. To characterize SC-Ad6 in vivo, groups of 5 BALB/c mice were injected intravenously with 3 1010 vp of the indicated viruses and livers were harvested on days one through four. qPCR of liver samples demonstrated that RD-Ad6 genome levels remained relatively constant over four days (Fig. 6A). In contrast, qPCR demonstrated that the genome copies of both SC-Ad6 and
C.M. Crosby, M.A. Barry / Virology 462-463 (2014) 158–165
161
Fig. 4. SC-Ad6 particles do not contain proteins VI, VII or IIIa. Infected 293-IIIa (A) and 293 (B) cells were separated on CsCl gradients. C) Purified virus particles were treated with SDS and run on a 10% SDS-PAGE, and stained with Bio-Safe Coomassie. D) Purified virus particles were treated with SDS and separated with agarose gel electrophoresis. A260/280 ratios for 293-empty: 1.04, 293-IIIa-empty: 1.11, 293-IIIa-mature: 1.26.
SC-Ad amplifies transgene expression in vivo in mice when compared to RD-Ad Liver samples that were collected for qPCR were also tested for luciferase activity (Fig. 6B). These data showed luciferase activity peaking at day 2 in the mice and declining through day 4. Consistent with genome amplification, SC-Ad and RC-Ad amplified transgene expressions above that mediated by the replicationdefective vector. In this case, SC-Ad6 and RC-Ad6 mediated 40and 300-fold higher luciferase activity than RD-Ad in the livers, respectively. SC-Ad and RC-Ad express more transgene product at lower multiplicities of infection than RD-Ad in primary human airway epithelia cells To determine how the vectors might behave in normal human cells, primary human small airway epithelial cells (HSAECs) were infected at varied multiplicities of infection with the GFPLuc expressing vectors and the cells were analyzed by microscopy 48 h later (Fig. 7). RD-Ad6 transduced most cells in the well at virus to cell ratios of 3.3 103 vp/cell. In contrast, both SC-Ad6 and RC-Ad6 generated at 33-fold lower multiplicities of infection. Fig. 5. Luciferase expression of Ad vectors in vitro. Human A549 (A) or mouse Hepa 1-6 (B) cells were infected at 1000 vp/cell. Luciferase expression was measured using Bright-Glo luciferase reagent at the indicated time points. Luminescence was measured using a Beckman Coulter plate reader.
RC-Ad6 were higher at all time points with an apparent peak at 2 days. At this peak, SC-Ad6 genome copies were 40-fold higher than RD-Ad6 while RC-Ad6 genomes were 125 times higher.
Discussion Human adenovirus vectors have shown potential for development as therapeutics for cancer, infectious disease, and genetic diseases. However, scale up into larger animals and humans and safety concerns have been significant hurdles for traditional replication defective and replication competent vectors. To overcome
162
C.M. Crosby, M.A. Barry / Virology 462-463 (2014) 158–165
Fig. 6. Genome amplification and luciferase expression in BALB/c mice. Groups of 30 female mice were injected with 3 1010 vp intravenously. At the indicated time points, liver samples were collected. (A) Total DNA was isolated and viral genomes were quantified by qPCR. N ¼ 15/virus group for day one, n¼ 5/virus group on days 2–4. (B) Samples were homogenized and Bright-Glo luciferase reagent was added at 1:1 ratio. Luminescence was measured using a Beckman Coulter plate reader. N ¼5/virus group/time point.
Fig. 7. GFP-mediated transduction of primary human airway epithelial cells. HSAECs were infected at the indicated vp/cell ratios and the monolayers were imaged on an inverted microscope 48 h later.
these challenges, we engineered “single cycle” replicating adenovirus 6 by deleting cement protein IIIa from the viral genome. SCAd6 effectively undergoes all of the early Ad life cycle events including early gene transcription and genome replication. The remaining late genes are transcribed and translated but in the absence of IIIa only empty, non-infectious virus particles are produced. This vector could have benefits over traditional replication defective and replication competent vectors. With RD-Ad6 infection, a low, but persistent level of luciferase protein is expressed both in vitro and in vivo. Due to the presence of E1 and other early genes, SC-Ad6 replicates its own genome along with any transgene it carries. In this proof of principle, SC-Ad6 produced significantly more luciferase than RD-Ad6 in vitro and in vivo in human and mouse cells. When the vectors were compared in human and mouse cells, the SC-Ad6 and RC-Ad6 were both able to replicate their genomes. This was interesting in part because it is generally accepted that adenoviruses do not replicate well in mice (Thomas et al., 2007). While it is true that infectious viruses are poorly replicated in mice, genome replication appears functional as demonstrated by qPCR. These data are consistent with early data showing that Ad5, another species C adenovirus, also amplifies in mouse liver after intravenous injection (Duncan et al., 1978). While the replicationcompetent Ad6 vectors did replicate their genomes and amplify transgene expression, these amplifications were approximately 50-fold less efficient than was observed in human cells. It should also be noted that luciferase expression in vitro and in vivo decreased after 2 days in mouse cells but not in human cells. It is unclear exactly why expression declined in mouse cells; however, this may be due to defects in the ability of human adenoviruses to complete the viral life cycle after genome replication. In particular, human Ads may fail to express late capsid components which may contribute to instability of newly synthesized viral genomes (Eggerding and Pierce, 1986). It is also possible that reduce late gene expression in mouse cells may perturb mouse cells and not humans cells to reduce expression. This is suggested in part by more rapid cell death in mouse Hepa1-6 cells than A549 cells even though SC- and RC-Ads replicate their genomes markedly higher in the human cells. From this, mice can be used to model single-cycle or replication-competent Ad6 but this model likely underestimates the impact of genome and transgene replication that may occur in non-human primates and humans. SC-Ad6 transgene expression was quite comparable with RCAd6 in human and mouse cell lines when tested in vitro. In contrast, in vivo, SC-Ad6 replication was 3-fold less efficient than RC-Ad6 and luciferase activity was 7-fold lower. These data suggest that factors present in vivo may modulate SC-Ad6 activity. These may be related to differences in genome packaging effects on innate immune responses or perhaps due to abortive second cycle infection by RC-Ad6 but not SC-Ad6. In contrast, when the vectors were tested in vitro on primary human airway cells, both SC-Ad and RC-Ad vectors generated markedly higher transduction at low multiplicities of infection. Notably, SC-Ad6 did not generate infectious virions making it appealing as safer replicating adenovirus vaccine than fully competent RC-Ads. While SC-Ad avoids a burst of infectious viruses from transduced cells, it does ultimately kill the initial target cells like RC-Ad. Therefore, these first infected cells are sacrificed to produce more transgene product. This makes SC-Ads less safe than RD-Ads but safer than RC-Ad6. This “Goldilocks” situation will require SC-Ads to be used under conditions that strike the right cost/benefit ratio. Sacrificing transduced cells is obviously not feasible for gene replacement therapy, so SC-Ads are not likely candidates for this type of therapy. In contrast, sacrificing mucosal cells for vaccines or tumor cells for cancer gene therapy may have merit.
C.M. Crosby, M.A. Barry / Virology 462-463 (2014) 158–165
In between these two extremes are using SC-Ads as gene-based vaccines, particularly for mucosal or oral vaccination. Fully wildtype RC-Ad4 and Ad7 adenoviruses have been used as oral vaccines to protect military personnel from respiratory infections. In this case, the vaccines must be delivered orally to avoid causing the respiratory disease one seeks to avoid (Couch et al., 1963). Oral RC-Ad delivery avoided the side effects of these wild viruses but still allowed mucosal vaccination to occur to protect from later RCAd respiratory infection. Therefore, RC-Ads applied to other sites run the real risk of causing adenovirus infections. In contrast, the SC-Ad vectors, can amplify antigen production during vaccination, but avoid the risk of adenovirus infection. They may particularly useful by the oral route, since most Ads are nearly quantitatively destroyed by stomach pH and digestive enzymes (Cheng et al., 2003). Therefore, RC-Ad4 and Ad7 vaccines were likely efficacious because they were able to amplify antigen production from the reduced number of cells that were infected by this stringent route. Proof of principle using RC-Ads safely in humans combined with the safer profile of SC-Ads, suggest these vectors may have utility also for systemic and mucosal vaccines against cancer and infectious agents.
Materials and methods Cell culture
163
amplified by PCR, adding BsaI sites on each end with compatible overhangs for MfeI. CMV-GFPLuc-SVA was inserted in pAd6PshSwa by direct ligation. pAd6-PshSwa-GL was linearized and recombined with pAd6-ΔE3. For RD-Ad generation, the E1 region was deleted by overlapping PCR of three fragments, one a 500bp region homologous to the region upstream of E1A, the second a blasticidin (BSD) resistance gene, and the third a 500 bp region homologous to pIX. The entire E1A/E1B region was removed by replacement with the BSD resistance gene by homologous recombination with pAd6-ΔE3-GL. For SC-Ad generation, the IIIa gene was deleted by overlapping PCR of three fragments, one a 500 bp region homologous to 52K, the second a zeocin resistance gene flanked by FRT sites, and the third a 500 bp region homologous to pII. The entire IIIa gene was replaced by the zeocin resistance gene, which was then removed with flp recombinase. E3 was deleted and GL was inserted by digesting pAd6-ΔE3-GL with PshAI and RsrII, and the E3 deletion and GL were recombined into pAd6ΔIIIa. Viruses were rescued in 293 cells or 293-IIIa cells and purified by double CsCl banding. Virus was desalted in 10% sucrose/KPBS. Virus particle concentration was determined by OD260. A260/280 ratios for each vector were: RD-Ad6-GL:1.3, SC-Ad6-GL:1.26, and RC-Ad6-GL:1.33. in vitro vector genome quantification 3 105 cells were plated in 6 well plates and infected at 1000 virus particles (vp)/cell. Two hours after infection, 1 ml of trypsin was added to cells. Detached cells were washed once with 5 ml of 5% FBS/DMEM. Cells were resuspended in 2 ml of 5% FBS/DMEM, and were plated in 6 well plates. After 24 h, cells were collected from plates with cell scrapers. Total DNA was isolated at 2–24 h after infection using the DNeasy Blood and Tissue kit according to manufacturer's protocol (Qiagen) with an RNase A digestion. Vector genomes were quantified using qPCR with primers against adenovirus hexon.
293 human embryonic kidney cells were purchased from Microbix (Toronto, Ontario, Canada). A549 lung carcinoma and mouse Hepa 1-6 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA). To produce 293-IIIa cells, the entire IIIa gene from Ad6 was PCR amplified and inserted into the plasmid pIRES-puro3 using BamHI and XhoI sites (Clontech, Mountain View, CA). The IIIa gene is expressed from a CMV promoter in a single transcript shared with an IRES-puromycin resistance gene. pIRES-puro3-IIIa was transfected in 293 cells using Polyfect reagent (Qiagen, Hilden, Germany), and cells were selected with 2 mg/ml puromycin. Cells were maintained as a mixed population, and IIIa expression was confirmed by western blot (data not shown). 293 cells were maintained in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS; HyClone, Rockford, IL) and penicillin/streptomycin at 100 U/mL (Gibco). Primary human small airway epithelial cells (HSAECs) were purchased from Lifeline Cell Technology (Frederick, MD). HSAECs were maintained in BronchiaLife SAE Complete Medium.
Concentration of DNA samples was determined by OD260 and diluted to 20 ng/μl. Real-time PCR was performed using the Applied Biosystems Prism 7900HT sequence detection system with SDS 2.3 software. Each well contained 10 μl Sybr Green (Applied Biosystems, Warrington, UK), 3.8 μl H2O, 0.6 μl of 10 μM F Primer, 0.6 μl of 10 μM R Primer, and 5 μl sample (i.e., 20ng DNA/ well).
Adenoviruses
Cell viability assay
A human adenovirus 6 plasmid was produced as described previously (Weaver et al., 2011), and genome modifications were completed using homologous recombination in bacteria as in Campos and Barry (2004). The E3 region of Ad6 was deleted by replacing the entire E3 region with a zeocin resistance gene. First, three PCR fragments were generated. The first was a 500 base pair product upstream of E3 and homologous to pVIII, the second a zeocin resistance gene flanked by short flippase recognition target (FRT) sites, and the third a 500 base pair product homologous downstream to gene U. A single DNA fragment was generated by overlapping PCR, which was recombined into pAd6. After recombination the zeocin gene was removed by flp recombinase in bacteria. A CMV-GFP–Luciferase-LZL-SV40 polyA cassette expressing a green fluorescence protein–luciferase fusion was inserted by first digesting pAd6 with PshAI and SwaI to make a smaller Ad6 plasmid with a unique MfeI site between the fiber and E4 open reading frames (pAd6-PshSwa). The CMV-GFPLuc-SVA gene was
Quantitative real time PCR (qPCR)
5 103 A549 cells were plated in 96 well plates and infected at 1000 vp/cell. At the indicated time points, 500 ml MTT (5 mg/ml) was added to the wells, and incubated 3.5 h at 37 1C. Media was removed and MTT developing reagent was added. After 15 min, A595 with A620 reference was read using the Beckman Coulter DTX 880 Multimode Detector system. in vitro virus burst assay 2 106 A549 cells were placed in a T75 flask and infected at 1000 vp/cell. 1 h post-infection, 3 mL trypsin was added to cells. Cells were washed once with 10 ml of 5% FBS/DMEM, and resuspended to a concentration of 2 105 cells/ml. 1 ml was collected and stored at 80 1C, and 1 ml was plated in each of 3 wells of a 6 well plate. Cells and supernatant were collected at indicated time points and stored at 80 1C. Cells were freezethawed 3 times and virus burst was quantified by TCID50 assay.
164
C.M. Crosby, M.A. Barry / Virology 462-463 (2014) 158–165
Transmission electron microscopy
Data Analysis
293 or 293-IIIa cells were infected with SC-Ad6 at 500 vp/cell. Cells were collected two days later and resuspended in 4% paraformaldehyde, 1% gluteraldehyde. Samples were processed and sectioned by the Electron Microscopy Core Facility (Mayo Clinic, Rochester, MN). Images were captured on a Philips CM10 Transmission Electron Microscope, equipped with a Gatan digital camera.
Graphs and statistical analyses were performed using Prism Graphical software.
SDS-PAGE and Coomassie blue staining 293 or 293-IIIa cells were infected with SC-Ad6 at 500 vp/cell. Cells were collected at two days, and virus was purified by single CsCl banding and desalted in 10% Sucrose/KPBS. Samples were boiled in 4x Laemelli's buffer, and run on a 10% tricine SDS-PAGE gel. The gel was stained with Bio-Safe Coomassie Stain (BioRad, Hercules, CA). Agarose gel electrophoresis of purified particles Equal numbers of purified virus particles were combined with 0.2% SDS at a Virus:PBS:SDS ratio of 1:1:2. Samples were combined with 6x DNA loading dye, and separated on a 0.8% agarose gel containing GelRed nucleic acid stain (Biotium, Hayward, CA). in vitro luciferase assay To quantify luciferase expression 1 103 cells were plated in black-walled 96-well plates and infected at 1000 vp/cell. At indicated time points, Bright-Glo luciferase reagent (Promega, Madison, WI) was added at a 1:1 ratio and luciferase activity was measured using the Beckman Coulter DTX 880 Multimode Detector system. Animals Female BALB/c mice were purchased from Harlan SpragueDawley (Indianapolis, IN). They were housed in the Mayo Clinic Animal Facility under the Association for Assessment and Accreditation of Laboratory Animal Care (AALAC) guidelines with animal use protocols approved by the Mayo Clinic Animal Use and Care Committee. All animal experiments were carried out according to the provisions of the Animal Welfare Act, PHS Animal Welfare Policy, the principles of the NIH Guide for the Care and Use of Laboratory Animals, and the policies and procedures of Mayo Clinic. Mice were injected intravenously with 3 1010 vp in 100 μL PBS. DNA isolation from mouse liver tissues Liver samples were collected and washed with PBS. Total DNA was isolated from liver samples using a Maxwell 16 DNA Purification Kit (Promega, Madison, WI). Luciferase expression of mouse liver tissues Liver samples were collected and washed with PBS. Samples were homogenized in T-Per Reagent containing protease inhibitors (Fisher Scientific, Rockford, IL). Homogenized tissue and Bright-Glo luciferase reagent (Promega, Madison, WI) were added at a 1:1 ratio of volume. Luciferase activity was measured using the Beckman Coulter DTX 880 Multimode Detector system.
Acknowledgments We would like to thank Mary Barry for excellent technical assistance and Tien Nguyen with assistance in processing liver samples. We also would like to thank Samuel Campos for originally generating the IIIa deletion construct. This work was supported by the Electron Microscopy Core Facility at Mayo Clinic. This work was supported by NIH/NIAID Grants R01 AI096967 and R01 CA136945.
References Abbink, P., Lemckert, A.A., Ewald, B.A., Lynch, D.M., Denholtz, M., Smits, S., Holterman, L., Damen, I., Vogels, R., Thorner, A.R., O'Brien, K.L., Carville, A., Mansfield, K.G., Goudsmit, J., Havenga, M.J., Barouch, D.H., 2007. Comparative seroprevalence and immunogenicity of six rare serotype recombinant adenovirus vaccine vectors from subgroups B and D. J. Virol. 81, 4654–4663. Campos, S.K., Barry, M.A., 2004. Rapid construction of capsid-modified adenoviral vectors through bacteriophage lambda red recombination. Hum. Gene Ther. 15, 1125–1130. Campos, S.K., Barry, M.A., 2007. Current advances and future challenges in Adenoviral vector biology and targeting. Curr. Gene Ther. 7, 189–204. Cheng, X., Ming, X., Croyle, M.A., 2003. PEGylated adenoviruses for gene delivery to the intestinal epithelium by the oral route. Pharm. Res. 20, 1444–1451. Couch, R.B., Chanock, R.M., Cate, T.R., Lang, D.J., Knight, V., Huebner, R.J., 1963. Immunization with types 4 and 7 adenovirus by selective infection of the intestinal tract. Am. Rev. Respir. Dis. 88 (Suppl.), 394–403. Demberg, T., Florese, R.H., Heath, M.J., Larsen, K., Kalisz, I., Kalyanaraman, V.S., Lee, E.M., Pal, R., Venzon, D., Grant, R., Patterson, L.J., Korioth-Schmitz, B., Buzby, A., Dombagoda, D., Montefiori, D.C., Letvin, N.L., Cafaro, A., Ensoli, B., RobertGuroff, M., 2007. A replication-competent adenovirus-human immunodeficiency virus (Ad-HIV) tat and Ad-HIV env priming/Tat and envelope protein boosting regimen elicits enhanced protective efficacy against simian/human immunodeficiency virus SHIV89.6P challenge in rhesus macaques. J. Virol. 81, 3414–3427. Demberg, T., Robert-Guroff, M., 2009. Mucosal immunity and protection against HIV/SIV infection: strategies and challenges for vaccine design. Int. Rev. Immunol. 28, 20–48. Duncan, S.J., Gordon, F.C., Gregory, D.W., McPhie, J.L., Postlethwaite, R., White, R., Willcox, H.N., 1978. Infection of mouse liver by human adenovirus type 5. J. Gen. Virol. 40, 45–61. Eggerding, F.A., Pierce, W.C., 1986. Molecular biology of adenovirus type 2 semipermissive infections. I. Viral growth and expression of viral replicative functions during restricted adenovirus infection. Virology 148, 97–113. Gomez-Roman, V.R., Florese, R.H., Patterson, L.J., Peng, B., Venzon, D., Aldrich, K., Robert-Guroff, M., 2006a. A simplified method for the rapid fluorometric assessment of antibody-dependent cell-mediated cytotoxicity. J. Immunol. Methods 308, 53–67. Gomez-Roman, V.R., Florese, R.H., Peng, B., Montefiori, D.C., Kalyanaraman, V.S., Venzon, D., Srivastava, I., Barnett, S.W., Robert-Guroff, M., 2006b. An adenovirus-based HIV subtype B prime/boost vaccine regimen elicits antibodies mediating broad antibody-dependent cellular cytotoxicity against nonsubtype B HIV strains. J. Acquir. Immune Defic. Syndr. 43, 270–277. Gomez-Roman, V.R., Grimes Jr., G.J., Potti, G.K., Peng, B., Demberg, T., Gravlin, L., Treece, J., Pal, R., Lee, E.M., Alvord, W.G., Markham, P.D., Robert-Guroff, M., 2006c. Oral delivery of replication-competent adenovirus vectors is well tolerated by SIV- and SHIV-infected rhesus macaques. Vaccine 24, 5064–5072. Hidajat, R., Xiao, P., Zhou, Q., Venzon, D., Summers, L.E., Kalyanaraman, V.S., Montefiori, D.C., Robert-Guroff, M., 2009. Correlation of vaccine-elicited systemic and mucosal non-neutralizing antibody activities with reduced acute viremia following intrarectal SIVmac251 challenge of rhesus macaques. J. Virol. 83 (2), 791–801. Liu, H., Jin, L., Koh, S.B., Atanasov, I., Schein, S., Wu, L., Zhou, Z.H., 2010. Atomic structure of human adenovirus by cryo-EM reveals interactions among protein networks. Science 329 (5995), 1038–1043. Malkevitch, N., Patterson, L.J., Aldrich, K., Richardson, E., Alvord, W.G., RobertGuroff, M., 2003. A replication competent adenovirus 5 host range mutantsimian immunodeficiency virus (SIV) recombinant priming/subunit protein boosting vaccine regimen induces broad, persistent SIV-specific cellular immunity to dominant and subdominant epitopes in Mamu-A*01 rhesus macaques. J. Immunol. 170, 4281–4289. Morgan, C., Marthas, M., Miller, C., Duerr, A., Cheng-Mayer, C., Desrosiers, R., Flores, J., Haigwood, N., Hu, S.L., Johnson, R.P., Lifson, J., Montefiori, D., Moore, J.,
C.M. Crosby, M.A. Barry / Virology 462-463 (2014) 158–165
Robert-Guroff, M., Robinson, H., Self, S., Corey, L., 2008. The use of nonhuman primate models in HIV vaccine development. PLoS Med. 5, e173. Nagayama, Y., Nakao, K., Mizuguchi, H., Hayakawa, T., Niwa, M., 2003. Enhanced antitumor effect of combined replicative adenovirus and nonreplicative adenovirus expressing interleukin-12 in an immunocompetent mouse model. Gene Ther. 10, 1400–1403. Patterson, L.J., Malkevitch, N., Venzon, D., Pinczewski, J., Gomez-Roman, V.R., Wang, L., Kalyanaraman, V.S., Markham, P.D., Robey, F.A., Robert-Guroff, M., 2004. Protection against mucosal simian immunodeficiency virus SIV(mac251) challenge by using replicating adenovirus-SIV multigene vaccine priming and subunit boosting. J. Virol. 78, 2212–2221. Peng, B., Voltan, R., Cristillo, A.D., Alvord, W.G., Davis-Warren, A., Zhou, Q., Murthy, K.K., Robert-Guroff, M., 2006. Replicating Ad-recombinants encoding nonmyristoylated rather than wild-type HIV Nef elicit enhanced cellular immunity. AIDS 20, 2149–2157. Peng, B., Wang, L.R., Gomez-Roman, V.R., Davis-Warren, A., Montefiori, D.C., Kalyanaraman, V.S., Venzon, D., Zhao, J., Kan, E., Rowell, T.J., Murthy, K.K., Srivastava, I., Barnett, S.W., Robert-Guroff, M., 2005. Replicating rather than nonreplicating adenovirus-human immunodeficiency virus recombinant vaccines are better at eliciting potent cellular immunity and priming high-titer antibodies. J. Virol. 79, 10200–10209. Reddy, V.S., Natchiar, S.K., Stewart, P.L., Nemerow, G.R., 2010. Crystal structure of human adenovirus at 3.5 A resolution. Science 329 (5995), 1071–1075. Stewart, P.L., Fuller, S.D., Burnett, R.M., 1993. Difference imaging of adenovirus: bridging the resolution gap between X-ray crystallography and electron microscopy. EMBO J. 12, 2589–2599.
165
Thomas, M.A., Spencer, J.F., Wold, W.S., 2007. Use of the Syrian hamster as an animal model for oncolytic adenovirus vectors. Methods Mol. Med. 130, 169–183. Weaver, E.A., Nehete, P.N., Buchl, S.S., Senac, J.S., Palmer, D., Ng, P., Sastry, K.J., Barry, M.A., 2009. Comparison of replication-competent, first generation, and helperdependent adenoviral vaccines. PloS One 4, e5059. Weaver, E.A., Nehete, P.N., Nehete, B.P., Yang, G., Buchl, S.J., Hanley, P.W., Palmer, D., Montefiori, D.C., Ferrari, G., Ng, P., Sastry, K.J., Barry, M.A., 2013. Comparison of systemic and mucosal immunization with helper-dependent adenoviruses for vaccination against mucosal challenge with SHIV. PloS One 8 (7), e67574, http://dx.doi.org/10.1371/journal.pone.0067574. Weaver, E.A., Palmer, D., Ng, P., Barry, M.A., 2011. Characterization of species C human adenovirus serotype 6 (Ad6). Virology 412 (1), 19–27, http://dx.doi.org/ 10.1016/j.virol.2010.10.041. Zhao, J., Lou, Y., Pinczewski, J., Malkevitch, N., Aldrich, K., Kalyanaraman, V.S., Venzon, D., Peng, B., Patterson, L.J., Edghill-Smith, Y., Woodward, R., Pavlakis, G. N., Robert-Guroff, M., 2003. Boosting of SIV-specific immune responses in rhesus macaques by repeated administration of Ad5hr-SIVenv/rev and Ad5hrSIVgag recombinants. Vaccine 21, 4022–4035. Zhao, J., Voltan, R., Peng, B., Davis-Warren, A., Kalyanaraman, V.S., Alvord, W.G., Aldrich, K., Bernasconi, D., Butto, S., Cafaro, A., Ensoli, B., Robert-Guroff, M., 2005. Enhanced cellular immunity to SIV Gag following co-administration of adenoviruses encoding wild-type or mutant HIV Tat and SIV Gag. Virology 342, 1–12.