Blood 8 , — Computational methods for prediction of T-cell epitopes—a framework for modelling, testing, and applications. Methods 34 4 , — Software tools for simultaneous data visualization and T-cell epitopes and disorder prediction in proteins. Synthetic peptides as antigens: pitfalls of conjugation methods.
Methods 78 1 , 59—69 The structure of proteins: two hydrogen-bonded helical configurations of the polypeptide chain. Natl Acad. USA 37 4 , — Rychlewski L, Fischer D. LiveBench the large-scale, continuous assessment of automated protein structure prediction.
Protein Sci. Pavlopoulou A, Michalopoulos I. State-of-the-art bioinformatics protein structure prediction tools. Shafiei, Kolsoom et al. Frequency of adenovirus serotype 8 in patients with keratoconjunctivitis, in Ahvaz, Iran. Evidence of molecular evolution driven by recombination events influencing tropism in a novel human adenovirus that causes epidemic keratoconjunctivitis.
Adenovirus Type 5 Hexon Protein
Novel human adenovirus causing nosocomial epidemic keratoconjunctivitis. Cloning, sequencing and bioinformatic analysis of hypervariable region of hexon gene of avian adenovirus 4 AAV4 associated with angara disease from Pakistan. Adenoviruses are common pathogens infecting animals and humans. They are classified based on serology, or genome sequence information. These methods have limitations due to lengthy procedures or lack of infectivity data. Adenoviruses are easy to produce and amenable to genetic and biochemical modifications, which makes them a powerful tool for biological studies, and clinical gene-delivery and vaccine applications.
Antibodies directed against adenoviral proteins are important diagnostic tools for virus identification in vivo and in vitro, and are used to elucidate infection mechanisms, often in combination with genomic sequencing and type specific information from hyper-variable regions of structural proteins. Here we describe a novel and readily useable method for cloning, expressing and purifying small fragments of hyper-variable regions of the adenoviral hexon protein.
In Western immunoblots with lysates from cells infected from thirteen human and three mouse viruses, these antibodies bound to homologous full-length hexon protein and revealed variable levels of cross-reactivity to heterologous hexons. Results from immuno-fluorescence and electron microscopy studies indicated that HAdV-B3 and MAdV-2 hexon antibodies recognized native forms of hexon.
The procedure described here can in principle be applied to any adenovirus for which genome sequence information is available. It provides a basis for generating novel type-specific tools in diagnostics and research, and extends beyond the commonly used anti-viral antibodies raised against purified viruses or subviral components. 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 ]. 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 , 5 , 6 , 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 , 13 , 14 , 15 ]. Most humans have been exposed to adenoviruses, and raise neutralizing antibodies and protective cytotoxic T cell responses [ 16 , 17 , 18 ]. In immune-competent individuals, AdVs cause only mild or no symptoms, with no observable long-term effects on health [ 19 ].
Anti-hexon Antibody Products | casadenonews.ga
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 , 22 , 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 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 , 26 , 27 , 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 , 32 , 33 ].
The serotype-specific residues recognized by these antibodies are located in seven hyper-variable regions HVRs protruding from the outer side of the viral capsid as deduced by comprehensive alignment of hexon sequences [ 34 , 35 , 36 , 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 , 39 , 40 , 41 , 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 ].
- Anti-hexon Antibody Products.
- Large-Scale Purification and Crystallization of Adenovirus Hexon | Springer Nature Experiments.
- Navigation menu.
- Identification of Adenovirus Serotype 5 Hexon Regions That Interact with Scavenger Receptors.
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 , 46 , 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 , 49 , 50 , 51 , 52 , 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 for production of specific hexon antibodies. Recombinant proteins generated in E. 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.
The cell lines were routinely screened for the absence of mycoplasma contamination.
- Hexon protein.
- The Portugal Journal (SUNY series, Issues in the Study of Religion);
- Additional information.
- Duplicate citations;
Shenk [ 56 ]. All other human prototype viruses were kindly provided by the late T. Hendrickx et al. All human wild type viruses were amplified in A cells and viral titers were determined by plaque assay using cells as described previously [ 54 ]. Studer manuscript in preparation. Hendrickx, manuscript in preparation.
Five grams of cell paste corresponding to a 4-l culture of E. Purified hexon HVRs fragments were used to immunize rabbits. The adjuvant was mixed with the antigen. Final bleeds were done following four immunizations. The obtained sera were mixed with Thimerosal at 0. The in vitro neutralization assay was performed as described in [ 59 ]. In brief, 2. Positive controls omitting the antibody and negative controls omitting the virus were included.
All tests were performed in triplicates and repeated twice. Staining and analyses by transmission microscopy TEM was performed as described earlier [ 65 ]. Grids were washed twice in PBS with 0. In the past, either purified virus or purified virus proteins were used for immunization to generate hexon-specific antibodies [ 44 , 66 ]. Since in our hands, purified MAdVs yields were lower compared to HAdVs and purification of MAdV hexon protein from lysates of infected cells was not efficient not shown , we decided to produce recombinant hexon fragments containing HVRs to induce specific antibodies.
All seven HVRs characteristic for the sequence diversity across different adenoviral sequences [ 34 , 35 , 36 , 37 ] coincided with the highest variable stretches in the MAdV sequences. Alignment of partial adenovirus hexon sequences and hexon protein trimer structure.
The color intensity indicates how highly an amino acid in a particular position is conserved amongst species. Highly conserved epitopes are highlighted in dark blue while variable epitopes are white or light blue.