Anti-hexon Antibody Research Articles

Structure-Based Modeling of Complement C4 Mediated Neutralization of Adenovirus.

Adenovirus (AdV) infection elicits a strong immune response with the production of neutralizing antibodies and opsonization by complement and coagulation factors. One anti-hexon neutralizing antibody, called 9C12, is known to activate the complement cascade, resulting in the deposition of complement component C4b on the capsid, and the neutralization of the virus. The mechanism of AdV neutralization by C4b is independent of downstream complement proteins and involves the blockage of the release of protein VI, which is required for viral escape from the endosome. To investigate the structural basis underlying how C4b blocks the uncoating of AdV, we built a model for the complex of human adenovirus type-5 (HAdV5) with 9C12, together with complement components C1 and C4b. This model positions C4b near the Arg-Gly-Asp (RGD) loops of the penton base. There are multiple amino acids in the RGD loop that might serve as covalent binding sites for the reactive thioester of C4b. Molecular dynamics simulations with a multimeric penton base and C4b indicated that stabilizing interactions may form between C4b and multiple RGD loops. We propose that C4b deposition on one RGD loop leads to the entanglement of C4b with additional RGD loops on the same penton base multimer and that this entanglement blocks AdV uncoating.

Development of a prototype immunochromatographic test for rapid diagnosis of respiratory adenovirus infection

Human adenoviruses comprise an important group of etiologic agents that are responsible for various diseases in adults and children, such as respiratory, ocular, gastroenteric, and urinary infections. In immunocompromised and organ-transplanted individuals, these agents can cause generalized infections. Rapid diagnostic methods for detecting these infectious agents are not widely available.The aim of this work was to produce monoclonal and polyclonal anti-adenovirus antibodies to be used in a rapid diagnostic test for respiratory infections.Adenovirus hexons were satisfactorily purified by ultracentrifugation and chromatography. After virus purification, anti-hexon monoclonal antibodies were produced and characterized, following classical methods. Antibodies were specific for adenoviruses 2, 3, 5, and 41. The proposed immunochromatographic test was standardized using colloidal gold.The standardization of the rapid test was sufficient to detect adenovirus antigens (in nasopharyngeal lavage samples) with sensitivity of 100% and specificity of 85% when compared to direct immunofluorescence.The immunochromatographic assay prototype was sufficiently sensitive to detect B (3), C (2 and 5), and F (41) adenovirus samples. Although based on preliminary data, the test demonstrated the same performance as direct immunofluorescence, but with the advantage of being a point-of-care test. Further studies are still needed to confirm its effectiveness in clinical practice.

Development of a quantitative immunocapture real-time PCR assay for detecting structurally intact adenoviral particles in water

Development of rapid, sensitive and specific methods for detection of infectious enteric viruses in water is challenging but crucial for gaining reliable information for risk assessment. An immunocapture real-time PCR (IC-qPCR) was designed to detect jointly the two major viral particle components, i.e. the capsid protein and the viral genome. Targeting both constituents helps circumventing the technical limits of cell culture approaches and the inability of PCR based methods to predict the infectious status. Two waterborne pathogenic virus models, human adenovirus types 2 and 41, were chosen for this study. IC-qPCR showed a detection limit of 10MPNCU/reaction with a dynamic range from 102 to 106MPNCU/reaction. Sensitivity was thus 100-fold higher compared to ELISA-based capture employing the same anti-hexon antibodies. After optimisation, application on environmental water samples was validated, and specificity towards the targeted virus types was obtained through the qPCR step. Heat-treated pure samples as well as surface water samples brought evidence that this method achieves detection of encapsidated viral genomes while excluding free viral genome amplification. As a consequence, adenovirus concentrations estimated by IC-qPCR were below those calculated by direct qPCR. The results demonstrate that the IC-qPCR method is a sensitive and rapid tool for detecting, in a single-tube assay, structurally intact and thus potentially infectious viral particles in environmental samples.

Directed Evolution of Mutator Adenoviruses Resistant to Antibody Neutralization

We incorporated a previously identified mutation that reduces the fidelity of the DNA polymerase into a human adenovirus vector. Using this mutator vector, we demonstrate rapid selection of resistance to a neutralizing anti-hexon monoclonal antibody due to a G434D mutation in hexon that precludes antibody binding. Since mutator adenoviruses can accumulate compound mutations that are unattainable using traditional random mutagenesis techniques, this approach will be valuable to the study of antivirals and host factor interactions.

Stepwise Loss of Fluorescent Core Protein V from Human Adenovirus during Entry into Cells

Human adenoviruses (Ads) replicate and assemble particles in the nucleus. They organize a linear double-strand DNA genome into a condensed core with about 180 nucleosomes, by the viral proteins VII (pVII), pX, and pV attaching the DNA to the capsid. Using reverse genetics, we generated a novel, nonconditionally replicating Ad reporter by inserting green fluorescent protein (GFP) at the amino terminus of pV. Purified Ad2-GFP-pV virions had an oversized complete genome and incorporated about 38 GFP-pV molecules per virion, which is about 25% of the pV levels in Ad2. GFP-pV cofractionated with the DNA core, like pV, and newly synthesized GFP-pV had a subcellular localization indistinguishable from that of pV, indicating that GFP-pV is a valid reporter for pV. Ad2-GFP-pV completed the replication cycle, although at lower yields than Ad2. Incoming GFP-pV (or pV) was not imported into the nucleus. Virions lost GFP-pV at two points during the infection process: at entry into the cytosol and at the nuclear pore complex, where capsids disassemble. Disassembled capsids, positive for the conformation-specific antihexon antibody R70, were devoid of GFP-pV. The loss of GFP-pV was reduced by the macrolide antibiotic leptomycin B (LMB), which blocks nuclear export and adenovirus attachment to the nuclear pore complex. LMB inhibited the appearance of R70 epitopes on Ad2 and Ad2-GFP-pV, indicating that the loss of GFP-pV from Ad2-GFP-pV is an authentic step in the adenovirus uncoating program. Ad2-GFP-pV is genetically complete and hence enables detailed analyses of infection and spreading dynamics in cells and model organisms or assessment of oncolytic adenoviral potential.

Adenovirus Transport via Direct Interaction of Cytoplasmic Dynein with the Viral Capsid Hexon Subunit

Early in infection, adenovirus travels to the nucleus as a naked capsid using the microtubule motor cytoplasmic dynein. How the dynein complex is recruited to viral cargo remains unclear. We find that cytoplasmic dynein and its associated proteins dynactin and NudE/NudEL, but not LIS1 or ZW10, colocalized with incoming, postendosomal adenovirus particles. However, in contrast to physiological cargos, dynein binding to adenovirus was independent of these dynein-associated proteins. Dynein itself directly interacted through its intermediate and light intermediate chains with the adenovirus capsid subunit hexon in a pH-dependent manner. Expression of hexon or injection of anti-hexon antibody inhibited virus transport but not physiological dynein function. These results identify hexon as a direct receptor for cytoplasmic dynein and demonstrate that hexon recruits dynein for transport to the nucleus by a mechanism distinct from that for physiological dynein cargo.

Complement Component C1q and Anti-hexon Antibody Mediate Adenovirus Infection of a CAR-Negative Cell Line

Realization of the potential clinical utility of recombinant adenovirus for gene therapy or vaccine development depends on a better understanding of the role of naturally occurring or therapy-induced anti-adenovirus antibodies. This study addresses the impact of anti-adenovirus neutralizing antibodies and the complement protein C1q on adenovirus infection of coxsackie and adenovirus receptor (CAR)-positive, and especially CAR-negative cells. Initially, transduction efficiency of adenovirus vectors was assessed in the presence or absence of human sera derived from healthy individuals that were seropositive for anti-adenovirus neutralizing antibodies. Infection was monitored by transgene expression in vitro using a replication-deficient adenovirus encoding green fluorescent protein (Ad-GFP). HeLa cells (CAR-positive) were readily infected by Ad-GFP and increasing concentrations of pooled sera increasingly inhibited infection. In contrast, rhabdomyosarcoma (RD) cells, a CAR-negative cell, were poorly infected by Ad-GFP. However, in the presence of human serum, robust GFP expression was observed. This expression was completely abrogated if the human serum was heat-inactivated. Addition of purified human C1q protein to the heat-inactivated serum restored GFP expression. Similar results were seen when human C1q protein was added to purified anti-hexon antibodies, but not to anti-fiber or anti-penton base antibodies, thus implicating anti-hexon antibodies as the infective antibody component of the human sera. These studies suggest that complement protein C1q and anti-hexon antibodies together can mediate efficient adenovirus infection in CAR-negative cell types.

Neutralizing Antibody Blocks Adenovirus Infection by Arresting Microtubule-Dependent Cytoplasmic Transport

Neutralizing antibodies are commonly elicited by viral infection. Most antibodies that have been characterized block early stages of virus entry that occur before membrane penetration, whereas inhibition of late stages in entry that occurs after membrane penetration has been poorly characterized. Here we provide evidence that the neutralizing antihexon monoclonal antibody 9C12 inhibits adenovirus infection by blocking microtubule-dependent translocation of the virus to the microtubule-organizing center following endosome penetration. These studies identify a previously undescribed mechanism by which neutralizing antibodies block virus infection, a situation that may be relevant for other nonenveloped viruses that use microtubule-dependent transport during cell entry.

Decreased immune reactivity towards a knobless, affibody-targeted adenovirus type 5 vector

In this study, a prototype Adenovirus type 5 (Ad5) vector deleted of the fiber knob domain and carrying an Affibody molecule as the targeting ligand showed decreased susceptibility to human pre-existing antibodies. This vector, Ad5/R7-Z(taq)Z(taq), has short fibers carrying seven shaft repeats, a non-native trimerization signal and an affibody molecule (Z(taq)) reactive to Taq polymerase. Ad5/R7-Z(taq)Z(taq) could be specifically targeted to 293 cells stably expressing membrane-bound anti-Z(taq) idiotypic affibody called Z(ztaq) (293Z(ztaq)). Sera from 50 blood donors were analyzed for neutralization activity (NA) against the parental Ad5/Fiwt vector and knobless Ad5/R7-Z(taq)Z(taq) on 293Z(ztaq) cells. Twenty-three sera had NA titers (> or =1:64) against Ad5/Fiwt (46%) and only two against Ad5/R7-Z(taq)Z(taq) (4%). Characterization of sera with NA titers showed that the knob domain is one of the targets of the antibodies. Neutralization assays using sera pre-adsorbed on knob and hexon proteins showed that the NA of the sera was carried mainly by anti-knob and anti-hexon antibodies, but in certain sera the anti-hexon antibodies represent the major population of the neutralizing antibodies (NAbs). Our results suggested that a combination of knob deletion and hexon switching could be an effective strategy for Ad vectors to better evade the anti-Ad NAbs.

Localized gene expression of axon guidance molecules in neuronal co-cultures

Axonal growth cones are guided to their targets by contact-dependent mechanisms or by diffusible chemotropic factors. Axon guidance by these factors typically involves culturing neurons on an acellular substrate which may not represent the in vivo biological environment. We developed two novel in vitro methods to create patterned gene expression of guidance molecules in a physiologically-relevant cellular environment. In the Matrigel™ assay, a droplet of adenovirus-Matrigel™ suspension was placed on astrocytes grown in Matrigel™. The adenovirus diffused through the gel and transduced underlying astrocytes, creating a radial infection gradient within a localized area. In the second model, recombinant adenovirus was bound to an anti-hexon antibody adsorbed onto stripe patterns of nitrocellulose. Once the cells were added, only those contacting the adenovirus were infected. The outgrowth pattern of chick DRG neurons on NGF, semaphorin 3A and brevican were studied. As expected, results showed robust axonal growth toward NGF as opposed to either secreted Sema 3A or membrane bound brevican, however subtle differences in axonal growth responses were observed in comparison to those obtained with less physiologically-relevant methods. Novel to this technology, the location and area of molecule expression can be controlled and manipulated in an intricate cellular environment.

Use of chimeric adenoviral vectors to assess capsid neutralization determinants

In order to elucidate the relative importance of neutralizing determinants on each of the three major adenoviral capsid components, we have generated chimeric vectors where the hexon protein, or the fiber protein, or both hexon and fiber proteins of one serotype (Simian Adenovirus 24/Pan 7) have been replaced by those of another (Simian Adenovirus 23/Pan 6). The effect of each replacement was evaluated by neutralization assays and by attempted vector re-administration into mice. Both hexon and fiber were found to harbor neutralization epitopes although in vivo transduction was more severely affected by anti-hexon antibodies.

Postentry Neutralization of Adenovirus Type 5 by an Antihexon Antibody

Antibodies against hexon, the major coat protein of adenovirus (Ad), are an important component of the neutralizing activity in serum from naturally infected humans and experimentally infected animals. The mechanisms by which antihexon antibodies neutralize the virus have not been defined. As a model system, murine monoclonal antibodies raised against Ad type 5 (Ad5) were screened for antihexon binding and neutralization activity; one monoclonal antibody, designated 9C12, was selected for further characterization. The minimum ratio of 9C12 to Ad5 required for neutralization was 240 antibody molecules per virus particle, or 1 antibody per hexon trimer. Analysis of antibody-virus complexes by dynamic light scattering and negative-stain electron microscopy (EM) showed that the virus particles were coated with electron-dense material but not aggregated at neutralizing ratios. Cryo-EM image reconstruction of the antibody-virus complex showed that the surface of the virus particle was covered by a meshwork of 9C12 antibody density, consistent with bivalent binding at multiple sites. Confocal analysis revealed that viral attachment, cell entry, and intracellular transport to the nuclear periphery still occur in the presence of neutralizing levels of 9C12. A model is presented for neutralization of Ad by an antihexon antibody in which the hexon capsid is cross-linked by antibodies, thus preventing virus uncoating and nuclear entry of viral DNA.

Targeting a Recombinant Adenovirus Vector to HCC Cells Using a Bifunctional Fab-Antibody Conjugate

We developed a specific adenoviral gene delivery system with monoclonal antibody (mAb) AF-20 that binds to a 180 kDa antigen highly expressed on human hepatocellular carcinoma (HCC) cells. A bifunctional Fab-antibody conjugate (2Hx-2-AF-20) was generated through AF-20 mAb crosslinkage to an anti-hexon antibody Fab fragment. Uptake of adenoviral particles and gene expression was examined in FOCUS HCC and NIH 3T3 cells by immunofluorescence; β-galactosidase expression levels were determined following competitive inhibition of adenoviral CAR receptor by excess fibre knob protein. The chimeric complex was rapidly internalized at 37°C, and enhanced levels of reporter gene expression was observed in AF-20 antigen positive HCC cells, but not in AF-20 antigen negative NIH 3T3 control cells. Targeting of recombinant adenoviral vectors to a tumor associated antigen by a bifunctional Fab-antibody conjugate is a promising approach to enhance specificity and efficiency of gene delivery to HCC.

Recombinant adenovirus gene transfer in adults with partial ornithine transcarbamylase deficiency (OTCD).

Human Gene TherapyVol. 10, No. 14 Recombinant Adenovirus Gene Transfer in Adults with Partial Ornithine Transcarbamylase Deficiency (OTCD)Mark L. Batshaw, James M. Wilson, Steven Raper, Marc Yudkoff, and Michael B. RobinsonMark L. BatshawSearch for more papers by this author, James M. WilsonSearch for more papers by this author, Steven RaperSearch for more papers by this author, Marc YudkoffSearch for more papers by this author, and Michael B. RobinsonSearch for more papers by this authorPublished Online:6 Jul 2004https://doi.org/10.1089/10430349950017068AboutSectionsPDF/EPUB ToolsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail FiguresReferencesRelatedDetailsCited byAbnormalities of Hepatic Protein MetabolismAdenoviral Vector-Based Vaccines and Gene Therapies: Current Status and Future Prospects13 March 2019Spliced: Boundary-work and the establishment of human gene therapy28 June 2017 | BioSocieties, Vol. 12, No. 2Adenoviruses7 March 2016The Ethics of Randomized Trials in Oncology25 November 2014Turning the Corner with Viral-based Gene Therapy—Development of the Rogue BiopharmaceuticalGene TherapyVirus-mediated gene delivery for human gene therapyJournal of Controlled Release, Vol. 161, No. 2Radioprotective gene therapy2 May 2011 | Expert Opinion on Biological Therapy, Vol. 11, No. 9The Death of Jesse Gelsinger: New Evidence of the Influence of Money and Prestige in Human Research1 June 2010 | American Journal of Law & Medicine, Vol. 36, No. 2-3Complement Component C1q and Anti-hexon Antibody Mediate Adenovirus Infection of a CAR-Negative Cell Line Van Tsai, Robin Varghese, Sundari Ravindran, Robert Ralston, and Gary Vellekamp30 December 2008 | Viral Immunology, Vol. 21, No. 4The gene delivery system for rheumatoid synoviumJapanese Journal of Clinical Immunology, Vol. 31, No. 1Gene therapy. 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Robinson.Recombinant Adenovirus Gene Transfer in Adults with Partial Ornithine Transcarbamylase Deficiency (OTCD).Human Gene Therapy.Sep 1999.2419-2437.http://doi.org/10.1089/10430349950017068Published in Volume: 10 Issue 14: July 6, 2004PDF download

Enhancement of calcium phosphate-mediated transfection by inclusion of adenovirus in coprecipitates.

Although coprecipitates of plasmid DNA and calcium phosphate (DNA:CaPi coprecipitates) were one of the first methods used for transfection of mammalian cells, they are inefficient for many cell types. Based on the recent finding that incorporating recombinant adenovirus in CaPi coprecipitates enhances expression of virus-encoded transgenes, we tested the hypothesis that including adenovirus in DNA:CaPi coprecipitates would increase the efficiency of transfection. We found that including adenovirus at the time of coprecipitate formation increased transgene expression in several cell types. Only a short incubation with cells was required, and the coprecipitates could be delivered in the presence of serum. Inclusion of adenovirus in coprecipitates did not increase DNA uptake by cells, and inactivated virus was also effective. Neutralizing anti-hexon antibody attenuated the enhancement produced by incorporating virus. These data suggest that the virus enhanced expression at a step after cellular uptake, probably by increasing DNA release from endosomes. The DNA:CaPi:Ad coprecipitates were at least as effective as complexes of DNA with adenovirus and polethylenimine or Lipofectin, but produced less cellular toxicity. The results suggest that DNA:CaPi:Ad coprecipitates have advantages for in vitro gene transfer and provide an attractive vehicle for investigating cellular mechanisms of gene transfer.

Rapid detection and serotyping of adenovirus by direct immunofluorescence

Four fluorescent antibody reagents were evaluated for their suitability for the identification of adenovirus isolates by immunofluorescence. The antibodies used in the reagents consist of monoclonal antibodies against adenovirus type 3 (Ad3), Ad4, Ad8, and adenoviruses of subgroup C (Ad1,2,5,6), serotypes known to occur in outbreaks of disease. Most of the monoclonal antibodies employed were reactive against type-specific antigens found on the hexon protein. Reagents employing two noncompeting anti-hexon antibodies were more sensitive than reagents prepared with only one monoclonal antibody, although both types of reagents exhibited a high degree of specificity. Five hundred and seventeen adenovirus isolates (359 of which had previously been typed by other methods) and 46 nonadenovirus isolates were examined with all four type-specific reagents in parallel with an adenovirus group-specific reagent. The results indicate that direct typing of adenovirus isolates is feasible, leading to significant savings in time compared to other typing methods and should contribute to the management of certain adenovirus infections, particularly during outbreaks.

Adenovirus protein-protein interactions: hexon and protein VI.

A variety of mastadenoviruses were denatured, their polypeptides separated by electrophoresis on SDS-polyacrylamide gels and transferred to nitrocellulose. The immobilized polypeptides were washed, incubated with buffers containing hexons from human adenoviruses (Ad) types 2, 5 and 12 and the location of bound hexons was detected with anti-hexon antibodies. It was found that hexons from any of the three human adenovirus types bound to protein VI from all the mastadenoviruses examined. Furthermore we found that hexon-VI binding was significantly greater than the interaction between hexon and the precursor to VI, pVI. This binding was susceptible to detergents and to changes in pH or salt concentration. A rabbit polyclonal antibody was raised against a recombinant protein derived from the middle third of pVI from Ad2 and was used to quantify the difference in binding and to demonstrate the presence of a single intermediate (designated iVI) in the processing of pVI to VI. The affinity between iVI and hexon was considerably greater in our assay than that of pVI but was less than that between hexon and VI. A complementary binding of recombinant iVI to immobilized hexons was also demonstrated. This latter interaction, however, was only observed when hexon preparations were not boiled prior to electrophoresis, substantiating the proposition that the recognition motif on the hexon was conformation-dependent. These results are discussed in the context of understanding further the molecular basis of protein-protein interactions between the structural proteins of adenoviruses and the factors involved in virion maturation.

A capture enzyme-linked immunosorbent assay for virus infectivity titrations as exemplified in an adenovirus system.

An enzyme-linked immunosorbent assay (ELISA), employing a capturing antihexon monoclonal antibody specifically recognizing free hexons, was developed for quantitative infectivity titration of adenovirus in a microscale titration assay. The method is based on the quantitative assessment of the total excess production of the major structural protein late in infection in samples consisting of 10(5) virus-infected HeLa cells maintained as stationary suspension cultures. Results are obtained with a coefficient of variation of 10% within 50 hours after virus infection. The method was designed for monitoring substances interfering with viral replication, e.g., neutralizing antibodies or antiviral drugs. Since it measured the total antigen content associated with cells as well as antigens possibly released into the growth medium the general approach should be applicable to any viral system where a structural protein is synthesized in excess.

Neutralization of adenoviruses: kinetics, stoichiometry, and mechanisms.

Kinetic curves for neutralization of adenovirus type 2 with anti-hexon serum revealed no lag periods even when the serum was highly diluted or when the temperature was lowered to 4 degrees C, thus indicating a single-hit mechanism. Multiplicity curves determined with anti-hexon serum displayed a linear correlation between the degree of neutralization and dilution of antiserum. Neutralization values experimentally obtained under steady-state conditions fully fitted a single-hit model based on Poisson calculations. Quantitation of the amount of 125I-labeled type-specific anti-hexon antibodies needed for full neutralization of adenovirus showed that 1.4 antibodies were attached per virion under such conditions. Virions already attached to HeLa cells at 4 degrees C were, to a large extent, neutralizable by anti-hexon serum, whereas anti-fiber and anti-penton base antisera were negative. It is suggested that adenovirus may be neutralized by two pathways: aggregation of the virions (extracellular neutralization) as performed by anti-fiber antibodies and blocking of virion entrance from the acidic endosomes into the cytoplasm (intracellular neutralization). The latter effect could be obtained by (i) covering of the penton bases, as performed by anti-penton base antibodies, thereby preventing interaction between the penton bases and the endosomal membrane, which results in trapping of virions within endosomes, and (ii) inhibition of the low-pH-induced conformational change of the viral capsid, which seems to occur in the endosomes and is necessary for proper exposure of the penton bases, as performed by anti-hexon antibodies.

Purification of adenovirus hexon by high performance liquid chromatography

Hexon is the major structural protein of adenovirus, and has significance in studies of virus structure and function, vaccine development, and immunodiagnosis. We describe a simple, single-step, anion-exchange high performance liquid chromatography (HPLC) method for the high yield purification of hexon. Purity of the isolated hexon was assessed by SDS-PAGE and HPLC methods. The isolated hexon was immunologically reactive with anti-hexon monoclonal antibody in a dot-blot assay. It also retained immunogenicity, as polyclonal antisera from rabbits immunized with hexon showed the desired antigen specificity. The enhanced speed of this purification method allows for the efficient isolation of hexon from various serotypes, and thus may facilitate comparative studies of hexon immunobiology.