Comparative thermal stabilities of recombinant adenoviruses and hexon protein

Differential scanning calorimetry was used to identify the thermal stability profile of the replication deficient and protein IX deleted recombinant adenovirus type 5 that contains the p53 transgene (rAd/p53) in phosphate buffered saline (vPBS) or 10% glycerol (TRIS/phosphate buffer). The wildtype adenovirus (Ad/WT) and purified hexon protein (major capsid protein) were also evaluated in 10% glycerol (TRIS/phosphate buffer) as controls. The thermal profile of rAd/p53 revealed three endothermic transitions (T1, T2 and T3) occurring between 25 degrees C and 90 degrees C. T1, which occurred at 46.7 degrees C in vPBS and 49.4 degrees C in TRIS/PO4 10% glycerol buffer, was irreversible following repeated scanning and attributed to the degradation of the intact vector. The latter two endothermic transitions, T2 and T3, occurring at 69 degrees C and 78 degrees C, respectively, corresponded with the two transitions of purified hexon in temperature and amount of heat absorbed. The thermal profile of Ad/WT revealed four endothermic transitions at 51.5 degrees C (T1), 70.5 degrees C (T2A), 73.6 degrees C (T2B), and 77.4 degrees C (T3). The higher temperature of degradation as well as additional transition was attributed to the presence of protein IX associated with the hexon. The positions and excess molar heat capacities of the intact rAds were found to be affected by pH, glycerol, vector concentration and the presence or absence of protein IX in the capsid. Irreversibility of T1 implied that the degradation of the intact virus may follow first-order kinetics. The thermal scan rate dependence of T1 further confirmed that degradation of the intact virus may be first-order. The apparent activation energies for the degradation of the intact vectors were determined from the scan rate dependence of T1 and shown to be affected by protein IX in the capsid and solution conditions. Analysis of rAd samples incubated at 45 degrees C by Field Emission Electron Microscopy (FESEM) confirmed that loss of single particles was first-order. Although aggregates were observed in the samples, degradation appeared to be the dominant reaction leading to disappearance of single virions from the aqueous matrix. Based on thermal and FESEM analysis, an empirical model was proposed that accounted for the disappearance of single rAd particles. At or near T1, degradation of rAd particles followed a unidirectional, pseudo-first order reaction. However, at lower temperatures, disappearance of single virions resulted from competing irreversible degradation and aggregation reactions.     

Hexon peptides of type 2, 3, and 5 adenoviruses and their relationship to hexon structure.

Peptides of hexons from type 2 and 5 (subgroup III) and type 3 (subgroup I) adenoviruses were produced by treatment with cyanogen bromide and were separated by isoelectric focusing in polyacrylamide gels containing 8 M urea. Peptides with identical isoelectric points, but from different hexon types, were considered to have structural similarities. According to this criterion for chemical relatedness, about two-thirds of the type 2 and 5 hexon peptides may be considered similar. In contrast, the majority of the type 3 hexon peptides differed chemically from peptides of type 2 and 5 hexons. Virions and free hexons were iodinated with 125-I in the presence of lactoperoxidase and H-2-O-2. When 125-I-labeled virions were disrupted and the hexon was purified, the highly labeled cyanogen bromide peptides were labeled. When purified hexons from the excess cellular pool were iodinated, peptides common to types 2, 3, and 5 (peptides 12 and 14) were most extensively labeled. Thus, hexons assembled in virions and those free in solution were iodinated differently. The data suggest that immunologically the hexons in viral capsids react differently from unassembled hexons because the polypeptide chains assume slightly different folding configurations in the two hexon forms and therefore expose different regions of the protein to antibodies.     

Structure-based high-throughput epitope analysis of hexon proteins in B and C species human adenoviruses (HAdVs)

Human adenoviruses (HAdVs) are the etiologic agent of many human infectious diseases. The existence of at least 54 different serotypes of HAdVs has resulted in difficulties in clinical diagnosis. Acute respiratory tract disease (ARD) caused by some serotypes from B and C species is particularly serious. Hexon, the main coat protein of HAdV, contains the major serotype-specific B cell epitopes; however, few studies have addressed epitope mapping in most HAdV serotypes. In this study, we utilized a novel and rapid method for the modeling of homologous proteins based on the phylogenetic tree of protein families and built three-dimensional (3D) models of hexon proteins in B and C species HAdVs. Based on refined hexon structures, we used reverse evolutionary trace (RET) bioinformatics analysis combined with a specially designed hexon epitope screening algorithm to achieve high-throughput epitope mapping of all 13 hexon proteins in B and C species HAdVs. This study has demonstrated that all of the epitopes from the 13 hexon proteins are located in the proteins' tower regions; however, the exact number, location, and size of the epitopes differ among the HAdV serotypes.     

Comparative sequence analysis of the hexon gene in the entire spectrum of human adenovirus serotypes: phylogenetic, taxonomic, and clinical implications

The adenovirus (AdV) hexon constitutes the major virus capsid protein. The epitopes located on the hexon protein are targets of neutralizing antibodies in vivo, serve in the recognition by cytotoxic T cells, and provide the basis for the classification of adenoviruses into the 51 serotypes known to date. We have sequenced the entire hexon gene from human serotypes with incomplete or no sequence information available (n = 34) and performed a comparative analysis of all sequences. The overall sequence divergence between the 51 human serotypes ranged from 0.7 to 25.4% at the protein level. The sequence information has been exploited to assess the phylogeny of the adenovirus family, and protein distances between the six AdV species (A to F) and among individual serotypes within each species were calculated. The analysis revealed that the differences among serotypes within individual species range from 0.3 to 5.4% in the conserved regions (765 amino acids [aa]) and from 1.5 to 59.6% in the variable regions (154 to 221 aa). Serotypes of different species showed an expectedly greater divergence both in the conserved (5.9 to 12.3%) and variable (49.0 to 74.7%) regions. Construction of a phylogenetic tree revealed three major clades comprising the species B+D+E, A+F, and C, respectively. For serotypes 50 and 51, the original assignment to species B and D, respectively, is not in accordance with the hexon DNA and protein sequence data, which placed serotype 50 within species D and serotype 51 within species B. Moreover, the hexon gene of serotype 16, a member of species B, was identified as the product of recombination between sequences of species B and E. In addition to providing a basis for improved molecular diagnostics and classification, the elucidation of the complete hexon gene sequence in all AdV serotypes yields information on putative epitopes for virus recognition, which may have important implications for future treatment strategies permitting efficient targeting of any AdV serotype.     

Future of adenoviruses in the gene therapy of arthritis

Recombinant adenoviruses are straightforward to produce at high titres, have a promiscuous host-range, and, because of their ability to infect nondividing cells, lend themselves to in vivo gene delivery. Such advantages have led to their widespread and successful use in preclinical studies of arthritis gene therapy. While adenoviral vectors are well suited to 'proof of principle' experiments in laboratory animals, there are several barriers to their use in human studies at this time. Transient transgene expression limits their application to strategies, such as synovial ablation, which do not require extended periods of gene expression. Moreover, there are strong immunological barriers to repeat dosing. In addition, safety concerns predicate local, rather than systemic, delivery of the virus. Continued engineering of the adenoviral genome is producing vectors with improved properties, which may eventually overcome these issues. Promising avenues include the development of 'gutted' vectors encoding no endogenous viral genes and of adenovirus–AAV chimeras. Whether these will offer advantages over existing vectors, which may already provide safe, long-term gene expression following in vivo delivery, remains to be seen.     

Unity and diversity in the human adenoviruses: exploiting alternative entry pathways for gene therapy

Human Ads (adenoviruses) have been extensively utilized for the development of vectors for gene transfer, as they infect many cell types and do not integrate their genome into host-cell chromosomes. In addition, they have been widely studied as cytolytic viruses, termed oncolytic adenoviruses in cancer therapy. Ads are non-enveloped viruses with a linear double-stranded DNA genome of 30-38?kb which encodes 30-40 genes. At least 52 human Ad serotypes have been identified and classified into seven species, A-G. The Ad capsid has icosahedral symmetry and is composed of 252 capsomers, of which 240 are located on the facets of the capsid and consist of a trimeric hexon protein and the remaining 12 capsomers, the pentons, are at the vertices and comprise the penton base and projecting fibre protein. The entry of Ads into human cells is a two-step process. In the first step, the fibre protein mediates a primary interaction with the cell, effectively tethering the virus particle to the cell surface via a cellular attachment protein. The penton base then interacts with cell-surface integrins, leading to virus internalization. This interaction of the fibre protein with a number of cell-surface molecules appears to be important in determining the tropism of adenoviruses. Ads from all species, except species B and certain serotypes of species D, utilize CAR (coxsackie and adenovirus receptor) as their primary cellular-attachment protein, whereas most species B Ads use CD46, a complement regulatory protein. Such species-specific differences, as well as adaptations or modifications of Ads required for applications in gene therapy, form the major focus of the present review.     

Hormone trajectories leading to human birth

The mechanisms regulating human parturition and labor remain unknown. This ignorance is expensive as preterm birth is responsible for 70% of neonatal mortality and 50% of cerebral palsy. Methods for the prediction of preterm birth and treatments for women in preterm labor have poor efficacy reflecting our limited knowledge of the mechanisms involved. Recent research has supported the view that parturition is a cascade of events that commences early in pregnancy and involves the mother, fetus, placenta, membranes, cervix and myometrium. Although a number of the key hormones and proteins involved have been identified, the relationships between these factors in time and tissues remain unclear. Placental production of Corticotropin-releasing hormone (CRH) is proposed as an early event regulating the cascade of events. Central to the onset of parturition will be a mechanism for progesterone withdrawal and estrogen activation in human. Two forms of progesterone receptor with opposing actions exist in the human myometrium. Progesterone receptor A (PR-A) is a dominant negative repressor of progesterone receptor B (PR-B). Preliminary studies strongly support the hypothesis that the onset of human parturition is initiated by rising concentrations of PR-A in the myometrium.     

Absence of a single repeat from the coding region of the human involucrin gene leading to RFLP.

The human involucrin gene has been mapped to the region q21-q22 of chromosome 1. Three of six Utah families examined were polymorphic for a PstI fragment of the involucrin gene. In one individual, the variant PstI fragment was found by DNA sequencing to be missing one of the 39 repeats that make up two-thirds of the coding region.     

Genetic analysis of a novel human adenovirus with a serologically unique hexon and a recombinant fiber gene

In February of 1996 a human adenovirus (formerly known as Ad-Cor-96-487) was isolated from the stool of an AIDS patient who presented with severe chronic diarrhea. To characterize this apparently novel pathogen of potential public health significance, the complete genome of this adenovirus was sequenced to elucidate its origin. Bioinformatic and phylogenetic analyses of this genome demonstrate that this virus, heretofore referred to as HAdV-D58, contains a novel hexon gene as well as a recombinant fiber gene. In addition, serological analysis demonstrated that HAdV-D58 has a different neutralization profile than all previously characterized HAdVs. Bootscan analysis of the HAdV-D58 fiber gene strongly suggests one recombination event.     

Immunochemical studies on human calcitonin M leading to information on the shape of the molecule

1. Two antisera were obtained from a single rabbit. Both are highly specific for human calcitonin M but react with different parts of the amino acid sequence. 2. The different sequences that react with the antibodies of the two antisera were located. The first antiserum reacts at two sites in the molecule, one in the sequence residues 11–18, probably with residue 17 as the immunodominant group, and another on either side of the 28–29 peptide bond. The second antiserum, harvested 9 months later, reacts principally at one site bridging the 28–29 peptide bond. 3. A consideration of the properties of the hormone's binding sites and of data relating biological activity to structure enables some conclusions to be drawn with regard to the shape of the molecule. It appears that the peptide chain is folded to bring N- and C-termini closer together and that there is non-covalent interaction between regions in the chain near both termini. One of these is located near residue 8.     

Virus chimeras for gene therapy, vaccination, and oncolysis: adenoviruses and beyond

Several challenges need to be addressed when developing viruses for clinical applications in gene therapy, vaccination, or viral oncolysis, including specific and efficient target cell transduction, virus delivery via the blood stream, and evasion of pre-existing immunity. With rising frequency, these goals are tackled by generating chimeric viruses containing nucleic acid fragments or proteins from two or more different viruses, thus combining different beneficial features of the parental viruses. These chimeras have boosted the development of virus-based treatment regimens for major inherited and acquired diseases, including cancer. Using adenoviruses as the paradigm and prominent examples from other virus families, we review the technological and functional advances in therapeutic virus chimera development and recent successful applications that can pave the way for future therapies.     

A method for predicting disease subtypes in presence of misclassification among training samples using gene expression: application to human breast cancer

MOTIVATION: An accurate diagnostic and prediction will not be achieved unless the disease subtype status for every training sample used in the supervised learning step is accurately known. Such an assumption requires the existence of a perfect tool for disease diagnostic and classification, which is seldom available in the majority of the cases. Thus, the supervised learning step has to be conducted with a statistical model that contemplates and handles potential mislabeling in the input data. RESULTS: A procedure for handling potential mislabeling among training samples in the prediction of disease subtypes using gene expression data was proposed. A real data-based simulation study about the estrogen receptor status (ER+/ER-) of breast cancer patients was conducted. The results demonstrated that when 1-4 training samples (N = 30) were artificially mislabeled, the proposed method was able not only in correcting the ER status of mislabeled training samples but also more importantly in predicting the ER status of validation samples as well as using 'true' training data.     

Association of endonuclease activity with serotypes belonging to the three subgroups of human adenoviruses.

Endonuclease activity has been detected in association with highly purified virions, pentons, and/or dodecons of adenovirus types 2, 3, 5, 9, 12, 15, and 16. Only single-strand scissions in substrate DNA were detected. The nuclease activity was detected by a highly sensitive ethidium bromide fluorimetric assay procedure.