Background
Adenoviruses (AdV) are ubiquitous pathogens, and affect vertebrates, including humans, livestock and wild animals. They undergo genetic recombination and periodically emerge in the human population, often in geographically distinct patterns [
1]. Human adenoviruses (HAdV) are comprised of ~60 types classified into seven species, A to G based on serology and DNA genome sequence [
2,
3]. The genetic information of adenoviruses is encoded in a double-stranded linear DNA molecule. It is imported into the cell nucleus upon stepwise disassembly of incoming virions [
4‐
7]. Adenovirus vectors are widely used in clinical gene therapy, mostly in cancer treatment and vaccination boost regimes [
8], in part due to strong innate immunity reactions due to viral danger signals [
9,
10]. Since the viral genome remains episomal throughout the infection cycle [
11], this gives rise to a robust, but transient viral gene expression. In addition, AdV vectors are widely used in clinical oncolytic applications due to the ease of genetic engineering and biochemical modifications [
12‐
15].
Most humans have been exposed to adenoviruses, and raise neutralizing antibodies and protective cytotoxic T cell responses [
16‐
18]. In immune-competent individuals, AdVs cause only mild or no symptoms, with no observable long-term effects on health [
19]. Persistent infections can occur [
20] which may be caused by interferon based anti-viral defense mechanisms [
21]. Adenoviruses can be live threatening to immune-compromised individuals [
21‐
23]. The high seroprevalence and immunogenicity of adenoviruses represents a considerable drawback when using these viruses as vectors for gene therapy, as pre-existing immunity leads to virus immune-complexes which can lead to inflammatory reactions [
13] and reduced treatment efficacy.
The AdV capsid is icosahedral, non-enveloped and consists of three major structural proteins fiber, penton base and hexon [
24]. The facets of the capsid are composed of 240 copies of the homotrimeric hexon protein, while 12 copies of the fiber protein are attached to pentameric penton protein bases on each of the vertices. Each hexon trimer has a pseudo-hexagonal base, which allows for close packaging within the facet, and three tower domains that are exposed on the exterior surface of the virion. All three major capsid proteins are immunogenic [
18,
25‐
28]. Occurrence of neutralizing antibodies against fiber protein in naturally infected individuals has been reported [
18,
25,
29,
30].
Importantly, the bulk of functionally significant neutralizing antibodies are directed against the hexon protein [
31‐
33]. The serotype-specific residues recognized by these antibodies are located in seven hyper-variable regions (HVRs 1-7) protruding from the outer side of the viral capsid as deduced by comprehensive alignment of hexon sequences [
34‐
37]. Experiments with hexon chimeric viruses, in which the complete hexon, single or combinations of HVRs were replaced by those of another serotype, revealed escape of humoral immune detection [
31,
32,
38‐
42]. Purified trimeric native hexon inhibited neutralization, but monomeric heat-denatured hexon did not, suggesting that neutralizing epitopes are complex and conformational but not linear [
27,
34]. In contrast to these findings, other groups reported generation of neutralizing antibodies using small HVR peptides only [
28,
43]. Non-neutralizing and cross-reactive hexon antibodies with common species- and genus-specific reactivity have been suggested to recognize sites that are accessible on the purified hexon but partially masked in the intact virion and outside of the HVRs [
27,
44]. Such antibodies were characterized using complement fixation, immunodiffusion, immunoprecipitation and immunoblot assays [
45‐
47].
Mouse adenoviruses (MAdV) represent an attractive model for studying viral and host factors involved in acute and persistent infections, testing oncolytic vectors in syngenic tumor models, and the development of anti-viral drugs [
48‐
53]. Like HAdVs they belong to the genus of Mastadenoviruses. Reagents such as antibodies against MAdV are scarce. Here we devised and tested the use of minimal hexon fragments containing the HVRs 1-6 for production of specific hexon antibodies. Recombinant proteins generated in
E. coli included hexon fragments derived from MAdV-1, −2, and HAdV-B3, −C5. Rabbit antibodies raised against MAdV-1, −2, and HAdV-3 fragments were tested in immunoblot assays, immunofluorescence, neutralization assays and EM for binding to denatured and native hexon. All three hexon antibodies recognized full-length hexon in Western immunoblots of lysates from infected cells, and two of three antibodies recognized native virus protein in immunofluorescence and transmission electron microscopy, but not in neutralization assays.
Methods
Cells and viruses
All cell lines including the human lung carcinoma cell line A549, the human cervical carcinoma cell line HeLa Ohio, the mouse colon carcinoma CMT93 and 3T6 fibroblast cells were grown in DMEM plus 8% FCS [
15,
54,
55]. The cell lines were routinely screened for the absence of mycoplasma contamination. Human HAdV-C5 wt300 was obtained from T. Shenk [
56]. All other human prototype viruses were kindly provided by the late T. Adrian (Medizinische Hochschule Hannover, Germany) and were verified by DNA restriction analysis [
57] and in part by hexon sequence analysis (R. Hendrickx et al., manuscript in preparation). All human wild type viruses were amplified in A549 cells and viral titers were determined by plaque assay using 911 cells as described previously [
54]. Recombinant E1/E3-deleted HAd-B3 and HAdV-C5 vectors expressing firefly luciferase were described previously [
58]. MAdV-1, −2 and −3 were kind gifts of K. Spindler (University of Michigan, Ann Arbor, USA), S. Compton (Yale University School of Medicine, USA) and D. Krüger (Charité Campus Mitte, Berlin Germany), respectively, and were amplified in CMT93 or 3T6 cells and titered by qPCR (R. Hendrickx et al., manuscript in preparation). The replication-competent HAdV-B3-pIX-FS2A-eGFP contains an eGFP open reading frame (ORF) genetically fused to the downstream end of the pIX gene using an autocleavage FS2A sequence ([
51] and L. Studer manuscript in preparation). Similarly, the replication-competent MAdV-1-E1A-FS2A-GL contains a
Gaussia luciferase (GL) ORF genetically fused to the downstream end of the E1A gene (R. Hendrickx, manuscript in preparation). The MAdV2-∆E1A-eGFP contains an E1A exon 1 deletion which was replaced by an eGFP cassette (R. Hendrickx, manuscript in preparation).
Construction of adenoviral hexon fragment expression vectors
For production of the four different hexon fragments the expression vector pGEX-6P-1 vector (GE Healthcare) was used. DNA fragments containing the hexon HVRs 1-6 were PCR-amplified using viral genomic DNA as template and inserted into the
EcoRI and
SalI cloning sites of pGEX-6P-1. The oligonucleotide sequences (Microsynth AG, Switzerland) used for cloning are listed in STab 1. Hexon HVRs 1-6 protein sequences, and relative molecular sizes of the GST-hexon and final hexon fragments are summarized in Additional file
1: Figure S1 and Additional file
2: Table S1.
Purification of adenoviral hexon protein fragments, production of rabbit polyclonal antibodies
Five grams of cell paste corresponding to a 4-l culture of E.coli BL21-CodonPlus(DE3)-RIPL cells, induced at 30 °C for 4 h with 0.3 mM IPTG, were resuspended in 25 ml of buffer T + 300 mM KCl (25 mM Tris-HCl, 10% glycerol, protease inhibitors (aprotinin, leupeptin, pepstatin A, PMSF), 0.5 mM EDTA, 1 mM dithiothreitol, 0.01% Nonidet-P40, pH 7.5). The cells were then disrupted by sonication and the lysate was clarified using ultracentrifugation (45 min, 4 °C, 100,000 × g).
The lysate was incubated with 1 ml of Glutathione-Sepharose beads (GE Healthcare) equilibrated with buffer T + 300 mM KCl for 1 h at 4 °C. The beads were then spun down and washed twice with 10 ml of T + 300 mM KCl and once with T + 50 mM KCl, followed by repeated protein elution with 1.5 ml of T + 100 mM KCl containing 30 mM glutathione. Fractions were analyzed by SDS-PAGE, pooled and treated with 100 units of PreScission Protease (GE Healthcare) for 22 h at 4 °C.
The solution was diluted with buffer 1xT until conductivity reached T + 50 mM KCl and was loaded on a 1 ml Mono Q column (GE Healthcare) equilibrated in T + 50 mM KCl. The protein was eluted using an 8 ml gradient of 50 – 500 mM KCl in buffer T.
Peak fractions were pooled, and loaded on a 5 ml Q5 Bio-Scale column (Bio-Rad) equilibrated in T + 50 mM KCl following adjustment of conductivity. The protein was eluted using a 100 ml gradient of 50 – 600 mM KCl in buffer 1xT. The elution times varied between different fragments but under these conditions, all of the fragments bound to the column, with HAdV-B3 eluting between 150 and 210 mM KCl, HAdV-C5 eluting between 270 and 350 mM KCl, MAdV-1 eluting between 90 and 130 mM KCl and MAdV-2 eluting between 60 and 110 mM KCl. Peak fractions were analyzed by SDS-PAGE, pooled and briefly incubated with a small amount of Glutathione-Sepharose beads to bind residual GST. The proteins were then concentrated using a 15 ml Vivaspin Turbo 15 concentration column with 10 kDa cut-off (Sartorius Stedim Biotech). All purification steps were performed at 4 °C and the final purified proteins were stored in buffer x at −80 °C until further use.
Purified hexon HVRs 1-6 fragments were used to immunize rabbits. Antibody development was carried out at BioGenes GmbH (Berlin, Germany). The animals were intramuscularly immunized using BioGenes’ adjuvant. The adjuvant was mixed 3:1 with the antigen. Final bleeds were done following four immunizations. The obtained sera were mixed with Thimerosal at 0.02% as preservative.
Neutralization assay
The in vitro neutralization assay was performed as described in [
59]. In brief, 2.5 × 10
4 cells (3T6 for mouse viruses, A549 for human viruses) were seeded in 100 μl DMEM-FCS medium in 96 well plates and grown overnight at 37 °C. Serial five-fold dilutions or fixed dilutions of the hexon neutralizing antibody were prepared in 50 μl DMEM-FCS medium and mixed with 25 μl of either medium or increasing amounts of the hexon protein fragment. The mixes were incubated for 30 min at 37 °C. Subsequently, 25 μl of the reporter virus were added to the solutions and the mixes were incubated for another 30 min. Positive controls omitting the antibody and negative controls omitting the virus were included. The virus concentration was adjusted to a multiplicity of infection (MOI) of 5 in the case of the human viruses, whereas for MAdV-1-E1A-FS2A-GL 10
3 genome equivalent virus particles (geq vp) per cell were used. The solutions were subsequently added to the cells and incubated for 24 h at 37 °C. For measuring the luminescence signal, the medium was aspirated and replaced with 40 μl of SteadyGLO lysis / substrate (Promega). The plates were incubated for 10 min at room temperature with orbital shaking. Then, 20 μl from each well was transferred to a Greiner LumiTrac plate and analyzed using a Tecan Plate reader with luminescence unit. When using MAdV-1-E1A-FS2A-GL, the renilla luciferase assay system (Promega) was used containing the coelenterazin substrate. All tests were performed in triplicates and repeated twice. The dog HAdV-C5 neutralizing serum was kindly provided by Anja Ehrhardt [
60,
61]. The hyperimmune rabbit anti-HAdV-B3 was a kind gift from A. Kajon (Lovelace Respiratory Program, Albuquerque, USA).
PAGE and western blot
For analysis of viral proteins derived from infected cells, cells were lysed in NETN (10 mM Tris pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5% NP40) complemented with protease inhibitors (Mini-Complete, Roche). Analyses of cleared cell lysates and hexon fragments from bacteria were performed by polyacrylamide gel electrophoresis [
62] followed by Coomassie Blue or PageBlue™ (Thermo Scientific) staining, or by Western blotting of electrotransfered protein to Immobilon-P membranes as described previously [
63]. Membranes were saturated in TBS-T containing 5% dry milk and incubated with primary antibodies including the rabbit anti-hexon fragment antibodies produced here, the rabbit anti-pIIIa antibodies (kindly provided by P. Hearing, School of Medicine, Stony Brook, USA) [
64] (both 1:1000), and the mouse monoclonal anti-actin antibody (5A7 biorbyt UK), anti-GAPDH antibody (MA5-15738, Thermo Scientific), and anti-tubulin antibody (DM1A, Sigma) (all at 1 μg/ml). Incubation with secondary antibodies included HRP-conjugated donkey anti-rabbit IgG, sheep anti-mouse IgG (GE Healthcare, both 1:4000), and sheep anti-dog IgG (GE Healthcare) for 1 h. The immunoreactivity was determined using the Luminata Crescendo Western HRP substrate (Millipore) and scored using the ImageQuant LAS 4000 imager (GE Healthcare).
Immunofluorescence imaging
1 × 104 human A549 or mouse 3T6 cells were seeded in 50 μl of DMEM-FCS medium and were incubated at 37 °C for 3 h. The cells were then infected with virus using an MOI of 1 for HAdV-B3-pIX-FS2A-eGFP, or alternatively, 100 geq vp per cell for MAdV-1-ΔE1A-eGFP and MAdV-2-ΔE1A-eGFP. The replication-competent human virus gave rise to a strong GFP signals and cells were incubated for 1 day, whereas the mouse cells were incubated for 5 days, due to the weaker GFP signals produced by these viruses. The cells were fixed using 3% PFA for 15 min at room temperature. The cells were washed three times with PBS-N3 and quenched for 10 min at room temperature with PBS-N3 + 25 mM NH4Cl. The cells were washed again three times and blocked for 1 h at room temperature in PBS-N3 + 0.5% BSA. The cells were then incubated with primary antibodies diluted 1:500 in blocking buffer for one hour at 4 °C, washed three times and incubated with secondary antibodies for 1 h at room temperature. The cells were washed and 50 μl of PBS-N3 + DAPI was added to the wells at least 30 min prior to imaging. The cells were then imaged at a 20× magnification using a ImageXpress Micro XLS system.
Immunogold-labelled electron microscopy imaging
Staining and analyses by transmission microscopy (TEM) was performed as described earlier [
65]. In brief, 10 μl of concentrated CsCl-purified adenovirus solutions were pipetted onto glow discharged TEM mesh grids. Virus was left to adsorb for 1-2 min, excess buffer was dried using a filter paper and grids were washed four times for 1 min in 10 μl of PBS with 10% goat serum. Grids were then incubated in 10 μl of primary antibody diluted 1:50 in PBS with 1% goat serum for 30 min at room temperature. This was followed by additional washing four times for 1 min in PBS with 10% goat serum and then incubation with 10 μl of 1:50 diluted secondary colloidal 10 nm gold-conjugated antibodies for 30 min at room temperature. Grids were washed twice in PBS with 0.1% BSA, three times in PBS and twice with deionized water for 1 min each. Virus was counterstained with 3% uranyl acetate for 20-40 s and the excess staining solution was drained with filter paper. Grids were allowed to air dry and were then imaged using a TEM FEI Tecnai G2 electron microscope. The 9C12 antibody included as primary antibody was developed by Laurence Fayadat and Wiebe Olijve, and was obtained from Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by the University of Iowa, Department of Biology, Iowa City, IA.
Discussion
Standard procedures to generate adenovirus hexon-specific antibodies include preparations of purified virions, or viral proteins for the purpose of immunization [
44,
66]. However, not all adenoviruses can be amplified efficiently in cell culture. For instance the two HAdVs belonging to species F (HAdV-F40 and −41) are notoriously difficult to grow to high titers [
70]. When trying to isolate MAdV-1 and-2 hexon protein from infected cell cultures using standard protocols [
71] we failed to obtain purified proteins. Since hexon is a large protein of 108 kDa that requires a viral chaperone activity [
72], production in
E.coli was found to be inefficient (unpublished data).
We decided to generate a small hexon subfragment consisting of about one fifth of the full-length size, but still containing six of the seven HVRs (Fig.
1a, Additional file
1: Figure S1 and Additional file
2: Table S1). HVR 7 was omitted since inclusion of this region increases the fragment size by about two-fold. In addition, neutralization profile analysis of hexon chimeric HAdV-C5 containing either HVRs 1-7 or only HVRs 1-6 of HAdV-C2 as well as epitope mapping of chimpanzee Ad68 hexon using a panel of neutralizing antibodies had suggested that most reactivity was contained in the HVRs 1-6 sequence [
27,
38]. Similar data were also reported with HAdV-B3 and -B7 [
73,
74]. In contrast, mapping of the epitope recognized by the mouse monoclonal 9C12 anti-HAdV-C5 hexon antibody suggested also a contribution of HVR 7 to binding of this neutralizing hexon antibody [
59,
75].
Following efficient expression in bacteria, our three-step purification procedure of the hexon HVRs 1-6 fragments using standard biochemical methods resulted in >90% pure protein preparations based on SDS-PAGE analysis, with sufficient quantities for immunization and production of polyclonal antibodies in rabbits (Fig.
2). It was somewhat surprising to find that the HAdV-C5 fragment was almost completely blocking a dog-anti HAdV-C5 neutralizing activity although the fragment consisted only of HVRs 1-6 (Fig.
3). Hexon fragment proteins consisting of HVRs 1-7 have been used for similar blocking experiments [
42], as well as a HVR 5-derived small peptide of HAdV-C5 [
28]. However, blocking activity by peptides may be rather exceptional, since it has been estimated that only a small fraction of <10% of the antibodies directed against native antigens bind to linear epitopes [
76]. In the case of HAdV-D48 hexon, neutralizing antibodies did not recognize linear peptides [
34]. Whether all of the four hexon fragments are capable of blocking neutralizing antibodies remains to be determined once the recombinant reporter viruses are available.
Hexon antigenic determinants and antibodies raised against hexon will not only be influenced by the nature of antigen used for immunization, e.g., purified virus, or hexon or partial fragment, but also by the immunization procedure which can include use of alum precipitation, adjuvants, and terminal boost with virus only. Thus, generation of mouse monoclonal antibodies resulted in cross-reactive genus-, species- or type-specific antibodies [
44,
66]. Type-specific and neutralizing epitopes have been suggested to be conformation dependent [
27,
34], whereas non-neutralizing and cross-reactive hexon antibodies with common species- and genus-specific reactivity have been suggested to recognize sites that are accessible on the purified hexon but partially masked in the intact virion and outside of the HVRs [
27,
44,
45].
A variable degree of cross-reactivity binding to heterologous hexons was observed when testing our three hexon Abs for binding in immunoblot assays, representing binding to mostly non-native forms of hexons. In the case of the HAdV-B3 hexon Ab, extensive cross-reactivity with most of the other tested hexons of the B1 and B2 subspecies viruses, and with the hexons of the D types were noticed (Fig.
4a). The observed cross-reactivity between species B and D is confirmed by phylogenetic tree analyses data of conserved and variable hexon regions, revealing that the B and D species viruses are more closely related than the B and C species viruses [
77]. The reduced cross-reactivity to hexon of HAdV-B16 is in line with the previously noted clustering of the HAdV-B16 hexon sequence to the HAdV-E4 sequence, suggested to have resulted from a recombination between sequences of these viruses [
77,
78]. Since the MAdVs clade is rather distant from the other Mastadenovirus clades [
48], it is not surprising to find lower levels of cross-reactivity of the MAdV-1 hexon antibodies and almost no cross-reactivity with the MAdV-2 hexon antibodies (Fig.
4b, c). The MAdV-1 hexon directed antibodies bound stronger to the MAdV-3 hexon than to the MAdV-2 hexon, reflecting the closer ancestor relationship of MAdV-1 and -3 compared to MAdV-2 [
48].
When used for detection of hexon proteins in IF of infected cells and for EM of purified virus, the HAdV-B3 and MAdV-2 hexon antibodies, but not the MAdV-1 antibody bound native hexon proteins (Figs.
5 and
6). However, when used for virus neutralizing assays, neither the HAdV-B3 nor the MAdV-1 antibodies revealed any blocking effect (Fig.
7), suggesting that the MAdV-1 antibody exclusively detects nonnative and denatured epitopes, whereas the HAdV-B3 antibody recognizes native hexon epitopes to some degree, but not those epitopes contained on trimeric hexons leading to neutralization.
Our results with the HAdV-B3 hexon antibody is in contrast to a study by Yuan et al. which generated HAdV-B3 neutralizing antibodies immunizing with various short HVR peptides bound to KLH carrier protein [
43]. However, the Yuan et al. study did not include other virus serotypes to confirm the specificity. Previous studies have shown that not all hexon binding antibodies exert neutralizing activity, but those that do bind with high affinity and function by rapid, single-hit kinetics, acting at a post-entry and TRIM21-dependent step [
18,
27,
79,
80]. Non-neutralizing antibodies were found to bind with lower avidity to native hexon than neutralizing antibodies [
27], which could explain why two of our three hexon antibodies still bound to intact virus in EM analysis, despite their lack of neutralization.
Acknowledgements
We thank Maarit Suomalainen for careful reading of the manuscript and Anja Ehrhardt (Ludwig Maximilians University of Munich, Germany) and A. Kajon (Lovelace Respiratory Program, Albuquerque, USA) for their generous gifts of neutralizing anti-HAdV-C5 and anti-HAdV-B3 antisera, respectively and P. Hearing (School of Medicine, Stony Brook, USA) for the rabbit anti-pIIIa serum. We also thank the Functional Genomics Centre Zurich for performing mass-spectrometry analysis and the Centre for Microscopy and Image Analysis, UZH for providing assistance with electron microscopy analysis.