Defective expression of mouse adenovirus Fl in human cells

HeLa cells infected with mouse adenovirus strain Fl ( AdFl ) produce at least 2000 times less virus than permissive mouse 3T3 cells. Viral DNA synthesis, however, proceeds unimpaired. The defect in virion production was linked to a dramatic reduction in the synthesis of AdFl structural proteins, in particular the hexon. The identity of the AdFl hexon gene product was recognized through its immunogenic reactivity towards an antiserum raised against the human adenovirus type 2 (Ad2) hexon gene product. This cross-reactivity is reflected in an extensive DNA sequence homology between AdFl and Ad2 DNA at position 53-60, the locus of the hexon gene, on the Ad2 physical map. Through hybridization at different formamide concentrations, the present study identifies one additional, highly conserved region at map positions 14-15 on the Ad2 genome.     

Immunogenicity of heterologous recombinant adenovirus prime-boost vaccine regimens is enhanced by circumventing vector cross-reactivity

The high prevalence of preexisting immunity to adenovirus serotype 5 (Ad5) in human populations has led to the development of recombinant adenovirus (rAd) vectors derived from rare Ad serotypes as vaccine candidates for human immunodeficiency virus type 1 and other pathogens. Vaccine vectors have been constructed from Ad subgroup B, including rAd11 and rAd35, as well as from Ad subgroup D, including rAd49. However, the optimal combination of vectors for heterologous rAd prime-boost vaccine regimens and the extent of cross-reactive vector-specific neutralizing antibodies (NAbs) remain poorly defined. We have shown previously that the closely related vectors rAd11 and rAd35 elicited low levels of cross-reactive NAbs. Here we show that these cross-reactive NAbs correlated with substantial sequence homology in the hexon hypervariable regions (HVRs) and suppressed the immunogenicity of heterologous rAd prime-boost regimens. In contrast, vectors with lower hexon HVR homology, such as rAd35 and rAd49, did not elicit detectable cross-reactive vector-specific NAbs. Consistent with these findings, rAd35-rAd49 vaccine regimens proved more immunogenic than both rAd35-rAd5 and rAd35-rAd11 regimens in mice with anti-Ad5 immunity. These data suggest that optimal heterologous rAd prime-boost regimens should include two vectors that are both rare in human populations to circumvent preexisting antivector immunity as well as sufficiently immunologically distinct to avoid cross-reactive antivector immunity.     

Determination of the nucleotide sequence for the penton-base gene of human adenovirus type 5

The major structural proteins of adenovirus (Ad), which form the external capsid, are hexon, penton base and fiber. The primary structure of the Ad5 penton base has been deduced from the nucleotide sequence of the corresponding gene. It has 98.6% homology with the sequence of the analogous protein from Ad2. This result is in contrast with the significantly lower homology found for the two other major structural proteins, the hexon and the fiber.     

Structural and immunochemical analysis of the products of proteolysis of simian adenovirus hexons

Hexon capsomers of simian adenovirus sim16 (SA7) and of human adenoviruses h5 (Ad5) and h6 (Ad6) were proteolytically digested and the resulting products studied by SDS-polyacrylamide gel electrophoresis and by radioimmunoprecipitation analysis. The trypsinolysis of native SA7 hexon leads to a stable molecular "core" containing 4-5 fragment species of 10 to 65 kDa and resembling the intact capsomer in quarternary structure (trimer). Similar cores but consisting of smaller fragments (less than 40 kDa) were obtained after chymotryptic digestion of native SA7, Ad5 and Ad6 hexons. The chymotryptic hexon fragments were also held together in pseudotrimeric structures. The similarity of proteolytic hexon fragment patterns between different primate adenoviral hexons suggested a homology to exist in localisation of the exposed tryptic and chymotryptic cleavage sites in their respective hexon polypeptide chains. Papain caused a complete hydrolysis of native SA7 hexon (trimer) yielding small peptides, but at first stage of digestion a stable papain hexon core containing small fragments (less than 10 kDa) was observed. The tryptic SA7 hexon cores in native state retained their antigenicity in reactions with homo- and heterologous antibodies, but after core denaturation the resulting fragments had no antigenic activity of native capsomer. In contrast to the data previously published, chymotryptic cores of SA7, Ad5 and Ad6 hexons not only reacted with respective homologous antibodies but also retained (at least in part) cross-reactive antigenic determinants. The questions of formation and stability of native adenoviral hexon conformation are discussed as well as the possible nature of hexon antigenic determinants.     

Characterization of two temperature-sensitive mutants of type 5 adenovirus with mutations in the 100,000-dalton protein gene.

Complementation analysis assigned the mutations of strains H5ts115 and H5ts116, two hexon-minus mutants, to the 100,000-dalton (100K) protein gene. Heterotypic marker rescue (i.e., type 5 adenovirus [Ad5] temperature-sensitive mutants DNA X EcoRI restriction fragments of Ad2 DNA) confirmed the results of previous marker rescue mapping studies, and the heterotypic recombinants yielded unique hybrid (Ad5-Ad2) 100K proteins which were intermediate in size between Ad5 and Ad2 proteins and appeared to be as functionally active as the wild-type 100K protein. Phenotypic characterization of these mutants showed that both the hexon polypeptides and the 100K polypeptides were unstable at the nonpermissive temperature, whereas fiber and penton were not degraded, and that the 100K protein made at 39.5 degrees C could not be utilized after a shift to the permissive temperature (32 degrees C). The role of the 100K protein in the assembly of the hexon trimer was also examined by in vitro protein synthesis. Normally, hexon polypeptides synthesized during an in vitro reaction are assembled into immunoreactive hexons. However, this assembly was inhibited by preincubation of the cell extract with anti-100K immunoglobulin G; neither anti-fiber immunoglobulin G nor normal rabbit immunoglobulin G inhibited hexon assembly. It is postulated that an interaction between the 100K protein and hexon polypeptides is required for effective assembly of hexon trimers.     

Restriction mapping and molecular cloning of adenovirus type 4 (subgroup E) DNA

The locations of thirty restriction endonuclease cleavage sites were determined on the genome of adenovirus type 4 (Ad4), the sole member of the subgroup E adenovirions. The restriction endonucleases BglII, EcoRI, HindIII, HpaI, KpnI, SalI, and XbaI cut Ad4 DNA 10, 3, 2, 3, 5, 5 and 3 times, respectively. Orientation of the linear Ad4 map with respect to left and right molecular ends was accomplished by taking advantage of the limited sequence homology between Ad2 and Ad4. Ten non-overlapping fragments of Ad4 DNA representing 98% of the genome, map units 1.6 to 99.6, have been cloned into the plasmid vector pKC7.     

Physical organization of subgroup B human adenovirus genomes.

Cleavage sites of nine bacterial restriction endonucleases were mapped in the DNA of adenovirus type 3 (Ad3) and Ad7, representative serotypes of the "weakly oncogenic" subgroup B human adenoviruses. Of 94 sites mapped, 82 were common to both serotypes, in accord with the high overall sequence homology of DNA among members of the same subgroups. Of the sites in Ad3 and Ad7 DNA, fewer than 20% corresponded to mapped restriction sites in the DNA of Ad2 or Ad5. The latter serotypes represent the "nononcogenic" subgroup C, having only 10 to 20% overall sequence homology with the DNA of subgroup B adenoviruses. Hybridization mapping of viral mRNA from Ad7-infected cells resulted in a complex physical map that was nearly identical to the map of early and late gene clusters in Ad2 DNA. Thus the DNA sequences of human adenoviruses of subgroups B and C have significantly diverged in the course of viral evolution, but the complex organization of the adenovirus genome has been rigidly conserved.     

In vitro construction of a recombinant adenovirus Ad2:Ad5

A hybrid virus containing the left half of the Ad5 genome and the right half of the Ad2 genome has been constructed by ligating together in vitro the BamHI.-A fragment of Ad5 (map co-ordinates 0–59.5) to the-SawHI-A fragment of Ad2 (map coordinates 59.5–100), and using this DNA to transfect susceptible cells. Viable progeny virus has been obtained which grows as well as the parental virus without any requirement for helper virus, and probably contains a hybrid hexon polypeptide consisting of the major part of the Ad5 hexon with an Ad2 carboxy terminus.     

Sequence of the DNA-binding protein gene of a human subgroup B adenovirus (type 7). Comparisons with subgroup C (type 5) and subgroup A (type 12)

The adenovirus type 7 (Ad7) single-stranded DNA-binding protein (DBP) structural gene has been sequenced and located between 66.7 and 62.3 map units. This region codes for a protein that contains 517 amino acid residues with a calculated molecular mass of 58,240 daltons. We compared the Ad7 amino acid sequence with those reported for the Ad5 (Kruijer, W., van Schaik, F.M.A., and Sussenbach, J.S. (1981) Nucleic Acids Res. 9, 4439-4457) and Ad12 (Kruijer, W., van Schaik, F.M.A., Speijer, J.G., and Sussenbach, J.S. (1983) Virology 128, 140-153) DNA-binding proteins. A greater amount of amino acid sequence homology was found in the carboxyl-terminal DNA-binding domain of the molecule. This homology is 61% between Ad7 and Ad5 and 49% when Ad12 was included in the comparison. The NH2-terminal domain of DBP retained a 49% homology between Ad7 and Ad5 and a 23% homology for all three serotypes. The greatest difference between the Ad7 and Ad5 DBPs is the absence, in the Ad7 protein, of 12 amino acids located between the two functional domains in the Ad5 protein (amino acids 151-162). In addition, three regions of high amino acid conservation between Ad5, Ad7, and Ad12 consisting of 9 (178-186), 9 (322-330), and 12 (464-475) consecutive amino acids (numbers refer to Ad5) in the DNA-binding portion of the molecule were revealed. These three regions contain a centrally located basic amino acid (183, 326, and 470) as well as an aromatic amino acid residue (181, 324, and 469). Since basic and aromatic amino acids have been implicated in other single-stranded DNA-binding protein/DNA interactions (Anderson, R.A., Nakashima, V., and Coleman, J.E. (1975) Biochemistry 14,907-917; Kowalczykowski, S.C., Lonberg, N., Newport, J.W., and von Hippel, P.H. (1981). J. Mol. Biol. 145, 75-104), these three conserved regions may represent DBP/DNA contact points.     

RGD inclusion in the hexon monomer provides adenovirus type 5-based vectors with a fiber knob-independent pathway for infection

Hypervariable region 5 (HVR5) is a hydrophilic, serotypically nonconserved loop of the hexon monomer which extrudes from the adenovirus (Ad) capsid. We have replaced the HVR5 sequence of Ad5 with that of heterologous peptides and studied their effects on virus viability and peptide accessibility. A poliovirus model epitope was first inserted in a series of nine "isogenic" viruses that differed in their flanking spacers. Whereas virus productivity was not profoundly altered by any of these modifications, immunoprecipitation experiments under nondenaturing conditions demonstrated that epitope recognition by its cognate monoclonal antibody (C3 MAb) was strongly linker dependent and correlated perfectly with the ability of C3 MAb to inhibit transgene delivery and expression. An alphav-specific ligand (DCRGDCF) was then inserted in a suitable linker context to investigate whether hexon-modified capsids would enhance the transduction of cells displaying limiting amounts of the virus attachment receptors. Interestingly, although hexon has never been implicated in Ad entry, the modified virus significantly increased the transduction of human vascular smooth muscle cells in vitro. Competition experiments with 293 cells saturated with recombinant knob further indicated that the hexon-modified virus could use an additional, knob-independent pathway for entry. We concluded that genetic engineering of the Ad5 hexon monomer constitutes a novel and feasible approach to equip the virus with additional targeting ligands.     

Specific properties of two enteric adenovirus 41 clones mapped within early region 1A.

Enteric adenovirus type 40 (Ad40) and Ad41 form the sixth subgenus of human adenoviruses. They are associated with infantile diarrhea but cannot be isolated in conventional cell cultures. The genome of the fastidious enteric Ad41 has been cloned, and the cleavage sites of the genome produced by restriction endonucleases BamHI, EcoRI, HpaI, NruI, PvuI, and SalI have been mapped. To develop useful hybridization methods for direct detection of adenoviruses, a restriction fragment library containing Ad41 DNA, with plasmid pBR322 as vector, has been constructed. Clones have been isolated which contain 8 of 10 possible BamHI fragments of Ad41, inserted into the BamHI cleavage site of the vector. Two of these clones are particularly useful for the detection of adenoviruses. One clone detects members of all human adenovirus subgenera, and the second clone is specific for enteric adenoviruses, in particular Ad41. A conspicuous absence of detectable homology was noted at 1.5 to 3.3 map units of the Ad41 genome in hybridizations against other serotypes of adenoviruses, including the closely related enteric Ad40. This sequence corresponds to the 5' portion of early region Ia.     

Structure and gene organization in the transformed Hind III-G fragment of Ad12

The nucleotide sequence of the transforming Hind III-G fragment of Ad12 DNA which encompasses the left 6.8% of the genome has been determined. The fragment was 2320 nucleotides long, and contained a GC cluster at positions 126-155 and a region extremely rich in AT at positions 1098-1142 (number from the leftmost end). Possible coding regions for the two transforming gene products were assigned. The predicted coding region for T antigen g is positions 502-1069 and positions 1144-1373, which are joined by splicing (266 amino acid residues, 30 kd), and that for T antigen f is positions 1845-2126 (94 amino acid residues, 10 kd). The sequence of the Hind III-G fragment was compared with that of the transforming DNA fragment of Ad5 which encompasses the left 8.0% of the genome (2809 nucleotides). There are several discrete regions with significant sequence homology. The comparison suggests that the regions in the left two thirds of the Ad5 and Ad12 transforming DNA fragments (map units 0-4.7% in Ad5 and 0-4.4% in Ad12) bear some resemblance in their gene organizations, and code for proteins containing structurally homologous regions.     

Cloning and sequencing of the bovine adenovirus type 2 early region 4

Bovine adenovirus type 2 (BAV2) is a medium size double-stranded DNA virus which infects both bovine and ovine species, resulting in mild respiratory and gastrointestinal disorders. To better understand the virus and its growth characterisitics in Madin-Darby bovine kidney (MDBK) cells, we have cloned and sequenced the extreme right-end segment of the BAV2 genome (90.5–100 map units). Analysis of the nucleotide sequence revealed 40 potential open reading frames (ORFs) with coding capacity for polypeptides that are 25 or more amino acid (aa) residues long. Six of these ORFs encode polypeptides that show homology to well-characterized early region 4 (E4) proteins of human adenovirus type 2 (Ad2) and Ad12. ORF1 has the potential to encode a 114 aa long polypeptide that is 54% homologous to the E4 14 kDa protein of Ad2. ORF2 encodes a 78 aa long polypeptide that exhibits 40% homology to the E4 13 kDa protein of Ad2. ORFs 3–6 encode polypeptides that have homology to the E4 34 kDa protein encoded by ORF6 of Ad2 and Ad12. ORFs 3, 4 and 5 encode 128, 96 and 31 aa long polypeptides, respectively. The 128-aa polypeptide exhibits 59% homology, while the 96 and 31 aa long polypeptides exhibit 61% and 70% homology to the E4 34 kDa protein, respectively. ORF6 has the potential to encode a 57 aa long polypeptide that has 67% homology to the E4 34 kDa protein of Ad2 and 50% homology to the E4 34 kDa protein of Ad12.     

Physical mapping of a large-plaque mutation of adenovirus type 2.

We have developed a simple method based on cotransfection of overlapping DNA restriction fragments for construction of recombinants of adenovirus type 2 (Ad2) and Ad5. When Ad2 DNA digested with restriction endonuclease EcoRI was cotransfected with Ad5 DNA digested with SalI, recombination occurred between Ad2 EcoRI-A (map position 0 to 59) and Ad5 SalI-A (map position 45 to 100). Analysis of the recombinant DNAs by digestion with EcoRI or BamHI restriction endonucleases indicated that, as expected, recombination had occurred in overlapping sequences (map position 45 to 59) between the Ad2 EcoRI-A fragment and the Ad5 SalI-A fragment. By using this method, several recombinants were constructed between a large-plaque (lp) mutant of Ad2 and wild-type Ad5. Cleavage of the recombinant genomes with restriction endonucleases BamHI, EcoRI, and HindIII revealed that the lp mutation is located within the left 41% of Ad2 genome.     

The 100K-chaperone protein from adenovirus serotype 2 (Subgroup C) assists in trimerization and nuclear localization of hexons from subgroups C and B adenoviruses

Recombinant hexons from subgroup C adenoviruses (Ad2 and Ad5) and from a member of subgroup B (Ad3) adenoviruses have been expressed in insect cells. When expressed alone, all three hexons were found to be insoluble and accumulated as inclusion bodies in the cytoplasm. However, co-expression of recombinant Ad2, Ad5 or Ad3 hexon with Ad2 L4-100K protein resulted in the formation of soluble trimeric hexons. EM analysis of hexons revealed that they were indistinguishable from native hexon capsomers isolated from Ad2-infected human cells, or released from partially disrupted adenovirions. This suggests that 100K acts as a chaperone for hexon folding and self-assembly into capsomer in insect cells. Since 100K protein assists in the trimerization of subgroup C hexon, and of subgroup B hexon protein, it implies that it functions in a manner that is both homo- and heterotypic. During the course of recombinant protein expression, the 100K protein was found in association with hexon monomers and trimers within the cytoplasm. In the nucleus, however, 100K was found in complexes with hexon trimers exclusively. EM observation of purified 100K protein samples showed a dumb-bell-shaped molecule compatible with a monomeric protein. EM analysis of hexon-100K protein complexes showed that interaction of hexon with the 100K protein occurred via one of the globular domains of the 100K protein molecule. Our data confirm the role of the 100K protein as a scaffold protein for hexon, and provide evidence suggesting its function in hexon nuclear import in insect cells.     

A pair of adjacent genes, cry5Ad and orf2-5Ad, encode the typical N- and C-terminal regions of a Cry5Adelta-endotoxin as two separate proteins in Bacillus thuringiensis strain L366.

A new DNA sequence cry5Ad/orf2-5Ad (GenBank accession number EF219060) was isolated from Bacillus thuringiensis strain L366. This DNA sequence contains two ORFs: cry5Ad (a previously unreported member of the cry5A gene family) and orf2-5Ad. cry5Ad is unique among cry5A genes in that it encodes only the N-terminal region of a typical Cry5Adelta-endotoxin. The cry5Ad sequence includes homology blocks 1-5, which are present in most B. thuringiensisdelta-endotoxins. The usual C-terminal region of a Cry5Adelta-endotoxin (including homology blocks 6-8) is encoded by orf2-5Ad. Both proteins encoded by cry5Ad and orf2-5Ad were found in IPTG-induced Escherichia coli, after a copy of cry5Ad/orf2-5Ad was cloned into the pQE32 expression vector and transformed into pREP4 E. coli cells. Both proteins were also found in parasporal crystal inclusions of B. thuringiensis L366. Sequencing of cDNA derived from transformed E. coli cells showed that the two ORFs are transcribed as a single mRNA. Extracts prepared from the recombinant E. coli expressing Cry5Ad and Orf2-5Ad were not toxic to nematode larvae (Haemonchus contortus), indicating that these two proteins are most likely not responsible for the nematocidal activity seen previously in the B. thuringiensis strain L366.     

HIV Antigen Incorporation within Adenovirus Hexon Hypervariable 2 for a Novel HIV Vaccine Approach

Adenoviral (Ad) vectors have been used for a variety of vaccine applications including cancer and infectious diseases. Traditionally, Ad-based vaccines are designed to express antigens through transgene expression of a given antigen. However, in some cases these conventional Ad-based vaccines have had sub-optimal clinical results. These sub-optimal results are attributed in part to pre-existing Ad serotype 5 (Ad5) immunity. In order to circumvent the need for antigen expression via transgene incorporation, the “antigen capsid-incorporation” strategy has been developed and used for Ad-based vaccine development in the context of a few diseases. This strategy embodies the incorporation of antigenic peptides within the capsid structure of viral vectors. The major capsid protein hexon has been utilized for these capsid incorporation strategies due to hexon''s natural role in the generation of anti-Ad immune response and its numerical representation within the Ad virion. Using this strategy, we have developed the means to incorporate heterologous peptide epitopes specifically within the major surface-exposed domains of the Ad capsid protein hexon. Our study herein focuses on generation of multivalent vaccine vectors presenting HIV antigens within the Ad capsid protein hexon, as well as expressing an HIV antigen as a transgene. These novel vectors utilize HVR2 as an incorporation site for a twenty-four amino acid region of the HIV membrane proximal ectodomain region (MPER), derived from HIV glycoprotein gp41 (gp41). Our study herein illustrates that our multivalent anti-HIV vectors elicit a cellular anti-HIV response. Furthermore, vaccinations with these vectors, which present HIV antigens at HVR2, elicit a HIV epitope-specific humoral immune response.     

Identification of a Nonstructural DNA-Binding Protein (DBP) as an Antigen with Diagnostic Potential for Human Adenovirus

Background

Human adenoviruses (HAdVs) have been implicated as important agents in a wide range of human illnesses. To date, 58 distinct HAdV serotypes have been identified and can be grouped into six species. For the immunological diagnosis of adenoviruses, the hexon protein, a structural protein, has been used. The potential of other HAdV proteins has not been fully addressed.

Methodology/Principal Findings

In this study, a nonstructural antigenic protein, the DNA binding protein (DBP) of human adenovirus 5 and 35 (Ad5, Ad35) - was identified using immunoproteomic technology. The expression of Ad5 and Ad35 DBP in insect cells could be detected by rhesus monkey serum antibodies and healthy adult human serum positive for Ad5 and Ad35. Recombinant DBPs elicited high titer antibodies in mice. Their conserved domain displayed immunological cross-reactions with heterologous DBP antibodies in Western blot assays. DBP-IgM ELISA showed higher sensitivity adenovirus IgM detection than the commercial Adenovirus IgM Human ELISA Kit. A Western blot method developed based on Ad5 DBP was highly consistent with (χ² =  44.9, P<0.01) the Western blot assay for the hexon protein in the detection of IgG, but proved even more sensitive.

Conclusions/Significance

The HAdV nonstructural protein DBP is an antigenic protein that could serve as an alternative common antigen for adenovirus diagnosis.