Immunological Basis of the Adenovirus 8-9 Cross-Reaction

The dedecon and hexon components of adenovirus types 8 and 9 have been extensively purified for use in establishing the basis of the cross-reaction between these types. Dodecons, the complete hemagglutinins, were purified 304- to 362-fold by fluorocarbon extraction, calcium phosphate batch chromatography, and ion-exchange column chromatography. Hexons, the group complement-fixation (CF) antigens, were purified 230- to 240-fold by erythrocyte adsorption, ion-exchange chromatography, and exclusion chromatography. Component antisera prepared in rabbits were tested in reciprocal fashion with crude virus and dodecon and hexon components. By hemagglutination-inhibition (HI), the dodecons of types 8 and 9 demonstrated the same predominantly one-sided relationship characteristic of the crude antigens. Some neutralizing activity was associated with both dodecons and hexons of each type. However, combining anti-dodecon and anti-hexon sera or producing antisera against the combined dodecon-hexon components resulted in neutralizing titers which were identical to titers obtained with antisera against the crude virus harvests. Dedecons of each type appear to share at least one antigenic determinant with hexons of the same type, and this determinant may reside on the vertex capsomere. Hexons possess group- and type-specific determinants, as shown by CF, neutralization, and immunodiffusion tests, and may exhibit some minor relationship between types 8 and 9. The results with the purified components are consistent with the predominantly one-sided antigenic relationship between types 8 and 9 in the conventional HI tests and the largely type-specific relationship by neutralization tests.     

Chimeric hexon HVRs protein reflects partial function of adenovirus

Adenovirus is widely used in gene therapy and vaccination as a viral vector, and its hypervariable regions (HVRs) on hexon are the main antigen recognition sites of adenovirus. The modification of this area by genetic engineering will change the antigenic specificity of the virus. In addition, recent studies have demonstrated the importance of coagulation factor X (FX) in adenovirus serotype 5-mediated liver transduction in vivo. The binding site of adenovirus to FX is the HVRs on hexon. By constructing five proteins containing chimeric HVRs from different adenovirus serotypes, we focused on the antigenic specificity and the affinity for FX of these proteins compared with the corresponding viruses. Our data showed that HVR5 and HVR7 had only a part of hexon activity to neutralizing antibodies (NAbs) compared with the complete activity of HVR1-7. Results also demonstrated a differential high-affinity interaction of the HVRs proteins with FX and indicated that HVRs protein had a similar binding ability with corresponding adenovirus serotype. These results highlighted some properties of chimeric HVRs proteins and revealed the influence on the structure and function of hexon proteins and adenovirus resulting from the HVRs.     

Generation and characterization of monoclonal antibodies to adenovirus.

Monoclonal antibodies (MoAbs) were generated following immunization of Balb/C mice with adenovirus type 5 grown in Hep2 cell line. Six clones reactive to hexon antigen of the virus were stabilized, of which 4 had mu-heavy chain specificity and 2 were of gamma-heavy chain type. Three of the clones (ADV-1, ADV-3 and ADV-5) had a high ELISA reactivity to the hexon antigen but exhibited differential specificity to the adenovirus types tested. In Western blotting, ADV-1 and ADV-3 reacted with all the adenovirus types tested (types 3,4,5,7 and 8) with reactions at 116 kDa region (hexon antigen), in addition, ADV-3 also had reactivity at 80 kDa region, the penton antigen. Reactivity to these adenoviral types by the 2 MoAbs was demonstrable by dot ELISA. ADV-5 had a type specific reaction only to adenovirus type 5 in dot ELISA with specificity in the hexon region in Western blotting. The reactivity of these 3 clones was not observed to the normal Hep2 tissue culture antigens and to the 3 enteroviruses tested (polio, coxsackie A9 and echo 4).     

Separation and Characterization of Soluble Adenovirus Type 9 Components

Four different soluble components of adenovirus type 9 (Rosen's group II) were identified. These were a complete hemagglutinin (HA), an incomplete HA, components carrying group-specific complement-fixing (CF) antigen, and components identified only by their hemagglutination-inhibition (HI) antibody consuming capacity and antigen activity in CF tests with an antiserum against complete HA. The complete HA sedimented relatively rapidly. It was composed of 12 pentons (vertex capsomers plus projections) aggregated into the form of a pentagonal dodecahedron. The length of the projections was about 12 to 13 mmu. Thus they appeared longer than the corresponding structures of types 3 and 11, but shorter than those of types 4 and 5. The rate of sedimentation of complete HA of type 9 was intermediate to those of the complete HA of types 3 and 11. The incomplete HA sedimented together with components carrying group-specific CF antigen, but could be separated from those by anion-exchange chromatography. Two different antigens were present in incomplete HA. One could absorb a group-specific hemagglutination-enhancing antibody, and was sensitive to treatment with trypsin. The other antigen could absorb the type-specific HI antibody and was not destroyed by trypsin. In addition to the incomplete HA, a separate population of more slowly sedimenting components showed a capacity to absorb HI antibody. These components could also be identified in CF tests when an antiserum against complete HA was applied. The incomplete HA, group-specific CF antigen, and slowly sedimenting HI antibody absorbing components are suggested to represent isolated penton, hexon, and fiber components, respectively.     

人3型腺病毒六邻体高变区HVR1、HVR2、HVR5、HVR7的氨基酸修饰限制研究

传统腺病毒载体的局限性使得外源抗原以衣壳融合的方式在腺病毒载体上的应用越来越广泛,但是在3型腺病毒（Adenovirus serotype 3, Ad3)载体六邻体高变区(Hypervariable region,HVR)改造过程中经常出现无法成功拯救病毒的情况,本研究主要根据对生物信息学预测的HVR1,HVR2,HVR5,HVR7中某些氨基酸进行删减或保留,通过构建重组Ad3载体pBRAdΔE3GFP-mHexon,转染AD293细胞,验证Ad3载体在六邻体高变区的这些氨基酸有所改动时对病毒拯救的影响。由此获得高变区HVR1、HVR2、HVR5和HVR7在基因工程改造中应该保留的氨基酸的数据,这一研究结果为人3型腺病毒六邻体融合表达策略提供了操作依据,也为人3型腺病毒六邻体表达外源抗原表位,作为多价疫苗载体展示平台的应用奠定了基础。     

Purification and properties of the hexon antigen of canine adenovirus serotype CAV-1

The main antigen and immunogen of canine adenovirus type 1 (CAV-1) has been purified to near homogeneity from cultural fluid of a CAV-1-infected primary cell culture by hydrophobic and anion-exchange chromatography. The hexon native form (trimer) was shown to be resistant against denaturation by SDS under conditions of SDS-PAGE performed without heating the samples. The monomer chain of the CAV-1 hexon was apparently identical in terms of electrophoretic mobility with that of the previously sequenced BAV-3 hexon polypeptide (103 kDA). In blot enzyme immunoassay only native trimers of CAV-1 hexon were detected by cross-specific polyclonal and monoclonal anti-hexon antibodies.     

Role of Adenovirus Structural Proteins in the Cessation of Host-Cell Biosynthetic Functions

Two of the adenovirus capsid proteins, the fiber and the hexon, complexed with either KB cell or type 5 adenovirus deoxyribonucleic acid (DNA). Maximal binding occurred at 0.01 m NaCl; increasing the ionic strength of the reaction mixture to 0.2 m NaCl resulted in a decrease in the association of either antigen to DNA. Variations of pH between 6.3 and 8.4 did not affect the binding of fiber antigen to DNA. Below pH 7.5, however, there was a small decrease in the ability of the hexon to bind nucleic acid. The association between the adenovirus structural proteins and DNA was reversible and was independent of whether the DNA was native or denatured. The fiber or hexon protein inhibited the DNA-dependent ribonucleic acid (RNA) polymerase and the DNA polymerase from KB cells. On a weight basis, the fiber protein inhibited enzymatic activity to a greater extent than the hexon. Increasing the template DNA concentration decreased this inhibition. The inhibition of the DNA-dependent RNA polymerase activity by either antigen could be reversed by increasing the ionic strength of the reaction mixture. After infection of KB cells with type 5 adenovirus, the levels of DNA and RNA polymerases remained unchanged for 15 to 20 hr. Thereafter, the specific activity of both enzymes decreased. By 30 hr postinfection, the polymerase activities were only about 30% of the enzyme activities in uninfected cells.     

Nanoporous crystals of chicken embryo lethal orphan (CELO) adenovirus major coat protein, hexon

CELO (chicken embryo lethal orphan) virus is an avian adenovirus that is being developed as a gene transfer vector. Its trimeric major coat protein (942 residues, 106,709 Da) has 42% sequence identity to human adenovirus type 2 (AdH2) hexon and 45% to AdH5 hexon. For structural studies, the growth of CELO virus has been optimized, and its hexon purified and crystallized. The hexon crystals, the first non-human example, diffract to 3.9 A resolution. Molecular replacement using the AdH5 model was used to identify the location of the CELO hexon within the unit cell. There is one hexon monomer in the asymmetric unit of the trigonal space group P321 (a=b=157.8 A, c=114.2 A, gamma=120 degrees) and the solvent content is 67.8%. The hexons pack in a hexagonal honeycomb so that large approximately 100 A diameter channels run through the entire crystal. This remarkable property of the crystals lends itself to their exploitation as a nanomaterial. Structural studies on CELO will elucidate the differences between avian and human adenoviruses and contribute to a better understanding of adenoviruses with non-human hosts.     

Soluble Components of Adenovirus Type 8

Soluble components of type 8 adenovirus have been studied. Four different components were isolated by anion-exchange chromatography and purified by further chromatographic procedures, by zonal centrifugation, and by erythrocyte absorption and elution. The four components exhibited the following characteristics. (i) Fiber antigen was trypsin-resistant and functioned as incomplete hemagglutinin (agglutinated rat and human erythrocytes only in the presence of certain types of adenovirus antisera). (ii) The penton was trypsin-sensitive, exerted a cytotoxic effect, and also showed incomplete hemagglutination, being active in the presence of a majority of heterotypic adenovirus antisera studied. (iii) The group-specific hexon antigen reacted in complement fixation reaction and gel precipitation with sera prepared against other types of adenoviruses, besides showing characteristics indicating the presence of a type-specific antigen component. (iv) The soluble complete hemagglutinin was trypsin-sensitive, displayed cytotoxic effect, adsorbed easily to human and rat erythrocytes, and could be eluted from them by means of receptor-destroying enzyme. The three hemagglutinins of adenovirus type 8 proved to be highly unstable, and their demonstration was only successful by using a large quantity of freshly prepared concentrated virus material. Considering these conditions, a method was developed for their concentration and purification.     

Paracrystal Formation in Cell Cultures Infected with Adenovirus Type 2

Large rod-shaped structures corresponding to paracrystals were seen in the nucleus, cytoplasm, or both of adenovirus type 2 (Ad2)-infected cells by immunofluorescence staining with antibody prepared against purified Ad2. In exception to this, Ad2-induced crystals did not stain with either hexon or fiber antibody. The crystalline structures were first observed in Ad2-infected Vero cells at 28 hr with a maximum number at 70 hr postinoculation. The kinetics of paracrystalline formation closely paralleled the experimental synthesis of infectious progeny virus. Acridine-orange staining revealed the lack of nucleic acids associated with the crystal. Also, the paracrystals stained intensely with phenanthrenequinone, suggesting that they are composed of basic proteins. Interferon induced by Newcastle disease virus from African green monkey kidney cell cultures was used to pretreat Vero cells prior to Ad2 infection. This resulted in inhibiting the formation of viral-induced paracrystals in 97% of the cells and reduced virus yields by 95%. The African green monkey kidney cell culture interferon did not reduce Ad2 yields in HeLa cell cultures or display any virus inhibitory activity in rabbit kidney cell cultures. Staining procedures, fluorescent-antibody tests with whole virus, hexon or fiber antibody, and interferon studies suggested that the paracrystals were viral-directed and composed of basic proteins (possibly core proteins).     

Characterization of a permissive epitope insertion site in adenovirus hexon

A robust immune response is generated against components of the adenovirus capsid. In particular, a potent and long-lived humoral response is elicited against the hexon protein. This is due to the efficient presentation of adenovirus capsid proteins to CD4+ T cells by antigen-presenting cells, in addition to the highly repetitive structure of the adenovirus capsids, which can efficiently stimulate B-cell proliferation. In the present study, we take advantage of this immune response by inserting epitopes against which an antibody response is desired into the adenovirus hexon. We use a B-cell epitope from Bacillus anthracis protective antigen (PA) as a model antigen to characterize hypervariable region 5 (HVR5) of hexon as a site for peptide insertion. We demonstrate that HVR5 can accommodate a peptide of up to 36 amino acids without adversely affecting virus infectivity, growth, or stability. Viruses containing chimeric hexons elicited antibodies against PA in mice, with total immunoglobulin G (IgG) titers reaching approximately 1 x 10(3) after two injections. The antibody response contained both IgG1 and IgG2a subtypes, suggesting that Th1 and Th2 immunity had been stimulated. Coinjection of wild-type adenovirus and a synthetic peptide from PA produced no detectable antibodies, indicating that incorporation of the epitope into the capsid was crucial for immune stimulation. Together, these results indicate that the adenovirus capsid is an efficient vehicle for presenting B-cell epitopes to the immune system, making this a useful approach for the design of epitope-based vaccines.     

Antigenic determinants of adenovirus capsids. I. Measurement of antibody cross-reactivity.

Evidence is presented that the type-specific antibody to the adenovirus hexon is not simply the antibody with the highest activity for cross-reactive determinants, but is a distinct, minority population that recognizes seperate determinants. To quantify it, we have developed an inhibition method with radio-immunoprecipitation (RIP) as a sensitive assay for the type-specific antibody that remains after all the excess of cross-reactive antibody has been blocked by heterologous antigen. During the primary response, 0.1 to 1% of antibody to types 2 or 5 hexon is type-specific, but after boosting, this population may reach 10 to 20%. Antibody to fiber is more than 70% type-specific during primary and secondary responses. The cross-reacting antibody can be removed on immunoabsorbent columns without affecting the virus neutralization titer of the serum.     

Comparative studies on the soluble proteins of adenovirus type 1

The soluble proteins of adenovirus type 1 have been separated and purified. Their antigenic characteristics were compared in different precipitation experiments performed in electric field. Both two-dimension immune electrophoresis and rocket electrophoresis can successfully be applied for quick diagnostic purposes. Quantitative determination of virus proteins is also feasible by rocket electrophoresis. The isoelectric point values of hexon, penton and fibre were pI 4.55, pI 4.69 and pI 7.07, respectively. The amino acid composition of type 1 adenovirus and its capsid components was determined from separated and purified protein preparations. The former differed in amino acid composition from the tissues used for virus propagation.     

Evidence that the penton base of adenovirus is involved in potentiation of toxicity of Pseudomonas exotoxin conjugated to epidermal growth factor.

When KB cells are incubated for 1 h with human adenovirus type 2 or type 5 (1 microgram/ml) and a conjugate of epidermal growth factor and Pseudomonas exotoxin (EGF-PE), protein synthesis is inhibited by 80 to 90%. Under these conditions, neither adenovirus nor EGF-PE alone has any effect on host protein synthesis. Thus, adenovirus enhances the toxicity of EGF-PE. A number of antibodies to intact virus and capsid components were tested for their ability to block the enhancing activity and virus uptake. At appropriate dilutions, antibodies prepared against intact virus and penton base blocked the enhancing activity without affecting virus uptake. Antibodies against hexon and fiber blocked virus uptake and enhancing activity in parallel. These studies suggest that the penton base is important in lysis of the vesicles which contain adenovirus and EGF-PE, and this base allows virus and toxin to enter the cytoplasm.     

Two- and three-dimensional hexon crystals.

Soluble hexon of type 1 adenovirus was highly purified with different techniques and dialysed against 0.5 M acetate buffer. With this procedure tetrahedral crystals were produced from the soluble hexon capsomers of the virus capsid. Electron microscopic observation of the crystallization process, revealed the development of dense two-dimensional "crystal sheets" following dialysis, in homogeneous hexon preparations containing single hexons. No such formations were observed so far with other types. The occurrence of two-dimensional crystals decreased proportionally to the appearance of three-dimensional crystals, which refers to their possible role in the mechanism of three-dimensional crystal formation.     

Characterization of a temperature-sensitive mutant of human adenovirus type 7.

The properties of a naturally occurring temperature-sensitive (ts) mutant of human adenovirus type 7 (Ad7) were studied. Mutant Ad7 (19), or E46-, was the nonhybrid adenovirus component derived from the defective simian virus 40 (SV40)-Ad7 hybrid (PARA). Growth of the mutant was restricted at 40.5 degrees C, and the ratios of virus yields in KB cells at 40.5 and 33 degrees C were 10(-2) to 10(-3). Viral DNA synthesis and the synthesis of adenovirus-specific antigens (tumor, capsid, hexon, and penton antigens) appeared normal at the restrictive temperature. The assembly of virus particles was aberrant, as determined by thin-section of infected cells. The infectivity of mutant virions was heat labile at 50 degrees C, suggesting a ts defect in a structural component of the viron. Analysis by polyacrylamide gel electrophoresis of [35S]methionine-labeled polypeptides synthesized in mutant-infected cells suggested that at least the major virion polypeptides were synthesized at the restrictive temperature. A lack of inhibition of host protein synthesis late in mutant infections, as compared with wild-type (WT) infections at both the permissive and nonpermissive temperatures, made quantitation of infected-cell polypeptides difficult. Analysis of the assembly of capsomeres from cytoplasmic extracts of infected cells on sucrose gradients and by non-dissociating polyacrylamide gel electrophoresis suggested that hexon capsomeres were made at 40.5 degrees C. The hexon capsomeres made by the mutant at either 33 or 40.5 degrees C displayed a decreased migration in the non-dissociating gels compared with the WT hexon capsomeres. The molecular weights of the mutant and WT hexon polypeptides were identical. These results suggest that the ts lesion of this group B human Ad7 mutant may be reflected in altered hexons. The mutant Ad7 interfered with the replication of adenovirus types 2 and 21 at the elevated temperature.     

Antigens of Pichinde virus I. Relationship of soluble antigens derived from infected BHK-21 cells to the structural components of the virion.

Antigens detected by the complement-fixation (CF) test were prepared from BHK-21 cells infected with Pichinde virus.The preparations contained two antigens demonstrable by immunodiffusion. The antigen present in abundance was heat stable, Pronase resistant, and had a molecular weight of 20,000 to 30,000 as estimated by gel filtration. Polyacrylamide gel electrophoresis of purified antigen demonstrated two low-molecular-weight polypeptides. An identical antigenic determinant was found by disrupting purified virus with Nonidet P-40; however, none of the viral polypeptides co-migrated with the polypeptides derived from purified CF antigen. Pronase digestion of disrupted virus did not alter antigenicity but degraded the viral peptides to sizes similar to those associated with the major CF antigen. These observations suggest that the major CF antigen of Pichnide virus is a cleavage product of the structural proteins of the virus.     

Nuclear import of adenovirus DNA in vitro involves the nuclear protein import pathway and hsc70

Adenovirus, a respiratory virus with a double-stranded DNA genome, replicates in the nuclei of mammalian cells. We have developed a cytosol-dependent in vitro assay utilizing adenovirus nucleocapsids to examine the requirements for adenovirus docking to the nuclear pore complex and for DNA import into the nucleus. Our assay reveals that adenovirus DNA import is blocked by a competitive excess of classical protein nuclear localization sequences and other inhibitors of nuclear protein import and indicates that this process is dependent on hsc70. Previous work revealed that the hexon (coat) protein of adenovirus is the only major protein on the surface of the adenovirus nucleocapsid that docks at the nuclear pore complex. This, together with our finding that in vitro nuclear import of hexon is inhibited by an excess of classical nuclear localization sequences, suggests a role for the hexon protein in adenovirus DNA import. However, recombinant transport factors that are sufficient for hexon import in permeabilized cells do not support DNA import, indicating that there are other as yet unidentified factors required for this process.     

Cryoelectron microscopy map of Atadenovirus reveals cross-genus structural differences from human adenovirus

A three-dimensional (3D) cryoelectron microscopy reconstruction of the prototype Atadenovirus (OAdV [an ovine adenovirus isolate]) showing information at a 10.6-A resolution (0.5 Fourier shell correlation) was derived by single-particle analysis. This is the first 3D structure solved for any adenovirus that is not a Mastadenovirus, allowing cross-genus comparisons between structures and the assignment of genus-specific capsid proteins. Viable OAdV mutants that lacked the genus-specific LH3 and p32k proteins in purified virions were also generated. Negatively stained 3D reconstructions of these mutants were used to identify the location of protein LH3 and infer that of p32k within the capsid. The key finding was that LH3 is a critical protein that holds the outer capsid of the virus together. In its absence, the outer viral capsid is unstable. LH3 is located in the same position among the hexon subunits as its protein IX equivalent from mastadenoviruses but sits on top of the hexon trimers, forming prominent "knobs" on the virion surface that visually distinguish OAdV from other known AdVs. Electron density was also assigned to hexon and penton subunits and to proteins IIIa and VIII. There was good correspondence between OAdV density and human AdV hexon structures, which also validated the significant differences that were observed between the penton base protein structures.     

Adenovirus type 2 terminal protein: purification and comparison of tryptic peptides with known adenovirus-coded proteins.

The protein covalently bound to the 5' termini of adenovirus type 2 DNA has been purified from virus labeled with [35S]methionine, using exclusion chromatography of disrupted virions to isolate the DNA-protein complex, which is then digested with DNase. The terminal protein isolated from mature virus is most effectively labeled if the cells are exposed to [35S]methionine during the "intermediate" period of 13 to 21 h postinfection, suggesting that the protein is synthesized during this interval. The tryptic peptides of the terminal protein were compared with those of several known adenovirus-coded proteins and found to be unrelated. In particular, the terminal protein is not related to the 38-50K early proteins encoded by the leftmost 4.4% of the adenovirus genome, one region essential for the transforming activity of the virus. Neither is it related to the 72K single-strand-specific DNA binding protein, the minor virion component IVa2, or the major capsid component hexon.