Adenovirus-mediated factor VIII gene expression results in attenuated anti-factor VIII-specific immunity in hemophilia A mice compared with factor VIII protein infusion.

Hemophilia A patients are typically treated by factor VIII (FVIII) protein replacement, an expensive therapy that induces FVIII-specific inhibitors in approximately 30% of patients with severe hemophilia. FVIII gene therapy has the potential to improve the current treatment protocols. In this report, we used a hemophilia A mouse model to compare the humoral and cellular immune responses between an E1/E2a/E3-deficient adenovirus expressing human FVIII directed by a liver-specific albumin promoter and purified recombinant FVIII protein infusion. Adenovirus-mediated FVIII expression did not elicit detectable CD4+ or CD8+ T cell responses and induced a weak antibody immune response to FVIII. In contrast, FVIII protein administration resulted in a potent anti-FVIII antibody response and moderate CD4+ T cell response. Furthermore, hemophiliac mice preimmunized with FVIII protein infusion to induce anti-FVIII immunity, and subsequently treated by adenovirus-mediated FVIII gene therapy, expressed therapeutic levels of FVIII despite the presence of low levels of anti-FVIII antibodies. No FVIII was detected in the plasma of mice with intermediate or high antibody levels, although anti-FVIII antibody levels in some vector-treated animals declined. The data support the hypothesis that liver-specific gene therapy-mediated expression of FVIII may be less immunogenic than traditional protein replacement therapy.

Optimization of bovine coronavirus hemagglutinin-estrase glycoprotein expression in E3 deleted bovine adenovirus-3.

Adenoviral vectors expressing foreign genes have many desirable properties in applications such as vaccination. Recently, we have generated replication-competent (E3 deleted) bovine adenovirus-3 (BAV-3) recombinants expressing significant amounts of glycoprotein D (gD) of bovine herpesvirus-1 (a DNA virus). However, attempts to express the RNA virus genes using the same strategy were not successful. In an effort to optimize the expression, we have constructed several BAV-3 recombinants carrying the hemagglutinin esterase (HE) gene of bovine coronavirus (BCV) in the E3 region with or without exogenous transcription control elements. The expression studies suggest that the introduction of a 137 bp chimeric intron upstream of the HE cDNA is able to increase the level of HE gene expression. The introduction of a SV40 early promoter or human cytomegalovirus (HCMV) immediate early (IE) promoter into the expression cassette changed the kinetics of the HE expression. However, the recombinant BAV-3 containing HE under the HCMV IE promoter replicated less efficiently than the wild-type BAV-3. These studies should prove useful in expression of other RNA viral genes in the E3 region of BAV-3 expression system.

The immunogenicity and efficacy of replication-defective and replication-competent bovine adenovirus-3 expressing bovine herpesvirus-1 glycoprotein gD in cattle.

Replication-competent and replication-defective bovine adenovirus type 3 recombinants expressing the bovine herpesvirus type 1 (BHV-1) glycoprotein D (gD) were tested for induction of gD specific immune responses in calves using intratracheal (1st and 2nd immunization) and sub-cutaneous (3rd immunization) route of immunization. The replication-defective recombinant BAV501 induced systemic immune responses against gD as low titers of anti gD-IgG were detected in the serum. However, the efficacy of the replication-competent BAV3.E3gD to induce gD-specific antibodies in the serum and the nasal secretions was superior to that of replication-defective BAV501 when both viruses were given at the same dosage. Partial protection from challenge was induced in calves immunized with replication-competent BAV3.E3gD. A dramatic increase in the titers of anti-gD IgG and IgA levels, both in serum and nasal secretions, following BHV-1 challenge (anamnestic response) suggested that the animals immunized with replication-defective BAV501 had been primed for gD-specific antibody responses.

In vivo dose threshold effect of adenovirus-mediated factor VIII gene therapy in hemophiliac mice.

While much is known about adenovirus biology from its development as a therapeutic gene delivery vehicle, an important question remains regarding the appropriate in vivo vector dose. We describe here an in vivo dose threshold effect with an adenoviral vector expressing human Factor VIII (FVIII) in hemophiliac mice. Upon administration of vector doses between 6 x 10(10) and 2 x 10(10) vector particles per mouse, FVIII was expressed linearly, whereas a dose of 1 x 10(10) vector particles per mouse did not result in detectable levels of FVIII activity. In contrast, in vitro transduction studies demonstrated linear transgene expression over 2 to 3 log units. To further define this dose threshold effect, a vector-mixing study was performed. Mice were injected with a total vector dose of 6 x 10(10) particles containing admixtures of FVIII vector plus a control vector lacking a transgene (null vector). With the admixture, FVIII activity was detected in mice that received 1 3 1010 particles of the FVIII vector, indicating that maintenance of the total viral input at 6 x 10(10) particles per mouse circumvented the threshold dose effect. This threshold dose effect could not be attributed to dose-dependent differences in liver toxicity nor to dose-dependent induction of cellular and humoral immune responses. Southern blot analysis of livers revealed that mice receiving the vector admixture contained FVIII DNA, accounting for the observed FVIII expression, whereas mice receiving 1 x 10(10) particles of FVIII vector had barely detectable FVIII DNA. These results suggest that the threshold effect is an in vivo phenomenon that will have important implications in defining the therapeutic window of adenoviral vectors for clinical applications.

Replication-defective bovine adenovirus type 3 as an expression vector.

Although recombinant human adenovirus (HAV)-based vectors offer several advantages for somatic gene therapy and vaccination over other viral vectors, it would be desirable to develop alternative vectors with prolonged expression and decreased toxicity. Toward this objective, a replication-defective bovine adenovirus type 3 (BAV-3) was developed as an expression vector. Bovine cell lines designated VIDO R2 (HAV-5 E1A/B-transformed fetal bovine retina cell [FBRC] line) and 6.93.9 (Madin-Darby bovine kidney [MDBK] cell line expressing E1 proteins) were developed and found to complement the E1A deletion in BAV-3. Replication-defective BAV-3 with a 1.7-kb deletion removing most of the E1A and E3 regions was constructed. This virus could be grown in VIDO R2 or 6.93.9 cells but not in FBRC or MDBK cells. The results demonstrated that the E1 region of HAV-5 has the capacity to transform bovine retina cells and that the E1A region of HAV-5 can complement that of BAV-3. A replication-defective BAV-3 vector expressing bovine herpesvirus type 1 glycoprotein D from the E1A region was made. A similar replication-defective vector expressing the hemagglutinin-esterase gene of bovine coronavirus from the E3 region was isolated. Although these viruses grew less efficiently than the replication-competent recombinant BAV-3 (E3 deleted), they are suitable for detailed studies with animals to evaluate the safety, duration of foreign gene expression, and ability to induce immune responses. In addition, a replication-competent recombinant BAV-3 expressing green fluorescent protein was constructed and used to evaluate the host range of BAV-3 under cell culture conditions. The development of bovine E1A-complementing cell lines and the generation of replication-defective BAV-3 vectors is a major technical advancement for defining the use of BAV-3 as vector for vaccination against diseases of cattle and somatic gene therapy in humans.

Transcription mapping and characterization of 284R and 121R proteins produced from early region 3 of bovine adenovirus type 3.

We established the transcription map of early region (E) 3 of bovine adenovirus 3 (BAV-3) by Northern blot, S1 nuclease protection assays, cDNA sequencing, and RT-PCR analysis. Five major classes of mRNAs were identified, which shared the 3' ends. Four classes of mRNAs transcribed from the E3 promoter also shared the 5' end, while one major class of mRNA transcribed from the major late promoter contained a tripartite leader sequence at the 5' end. These five transcripts have the potential to encode four proteins, namely 284R, 121R, 86R, and 82R. To identify the proteins, rabbit antiserum was prepared using a bacterial fusion protein encoding 284R or 121R protein. Serum against 284R immunoprecipitated protein of 26-32 kDa in in vitro translated and transcribed mRNA and three proteins of 48, 67, and 125 kDa from BAV-3-infected cells. Western blots and enzymatic digestions confirmed that the 284R protein is a glycoprotein, which contains only N-linked oligosaccharides, both high mannose (48 kDa) and complex types (67 kDa). Serum against 121R immunoprecipitated a protein of 14.5 kDa from in vitro translated and transcribed mRNA and BAV-3-infected cells. Although 121R protein shows limited sequence similarity to a 14.7-kDa protein of human adenovirus 5, the 284R protein appears to be unique to BAV-3. Since proteins encoded by the E3 region appear to influence adenovirus pathogenesis, the 284R protein may contribute to the unique pathogenic properties of BAV-3.

Development of porcine adenovirus-3 as an expression vector.

Porcine adenovirus-3 (PAV-3) was developed as an expression vector using homologous recombination in Escherichia coli BJ 5183. As a prerequisite, the complete genome of PAV-3 was first introduced as a PacI restriction fragment into a bacterial plasmid. The plasmid, when PacI restricted and transfected into swine testicular cells, produces an infectious virus. The potential of this procedure was demonstrated by the construction of several PAV-3 recombinants. Part of the E3 region, which is nonessential for virus replication under cell culture conditions, was identified and deleted from the virus genome. The gene for glycoprotein D (gD) of pseudorabies virus (PRV), which elicits PRV-neutralizing antibodies in pigs, was cloned and expressed from the E3 region of PAV-3. A 50 kDa polypeptide was identified in recombinant PAV-3-infected cell lysates by immunoprecipitation assays using gD-specific monoclonal antibodies. In another experiment, a region between the right inverted terminal repeat and the promoter of the E4 region was used to clone and express the chloramphenicol acetyltransferase (CAT) gene under the control of SV40 immediate early promoter. CAT gene expression was observed irrespective of the orientation of the CAT gene. These results indicate that the helper-independent recombinant PAV-3 could be used as an expression vector and has potential as a recombinant vaccine vector in pigs.

Characterization of early region 1 and pIX of bovine adenovirus-3.

The mRNAs from early region 1 (E1) and pIX of bovine adenovirus type 3 (BAV-3) have been studied by Northern blot, S1 nuclease, and cDNA analysis and transcriptional maps for the regions were constructed. The transcriptional map for the E1 region of BAV-3 is different from those of mouse and human adenoviruses for which transcriptional maps for the regions have been constructed. The E1A region of BAV-3 is located between 0.8 and 10.5 map units and several different transcripts are produced from the region using alternative splice donor sites. The transcripts from the E1A region overlap with those of E1B and pIX. In BAV-3, the E1B region maps between 4.2 and 10.5 map units and encodes two major mRNA species. The mRNAs of E1B region differ from each other in that the smaller mRNA coding for the 157R protein has a large intron removed from a region corresponding to the coding region of E1B 420R protein. As in HAVs, the E1B 420R protein of BAV-3 could be translated only by internal initiation from the larger bicistronic mRNA as there are no transcripts produced exclusively for the production of 420R protein. The transcriptional unit of pIX is transcribed from an independent promoter and encodes a structural component of the adenovirus capsid. To identify and characterize the proteins produced from the region, antibodies were raised in rabbits that recognized specific proteins in Western blot and immunoprecipitation assays.

Nucleotide sequence and transcription map of porcine adenovirus type 3.

The complete nucleotide sequence of porcine adenovirus type 3 was determined and a transcriptional map for the genome was constructed. The size of the genome is 34094 bp in length with an unusually high G + C content (63.7%), the highest thus far reported for any adenovirus. Overall organization of the genome is similar to that for previously sequenced adenoviral DNAs, but there also were distinct differences. The late regions genes are organized into six families, instead of five as they are in human adenovirus type 2. In contrast to bovine adenovirus type 3 and ovine adenovirus, which lack virion-associated RNA genes, the nucleotide sequence analysis of the viral genome indicates that it encodes one short VA RNA species. With the exception of the fiber and a 33-kDa nonstructural protein, the predicted amino acid sequences of the open reading frames in the late regions and the E2 region and IVa2 exhibited a high level of homology, whereas the deduced amino acid sequences of ORFs in E1, E3, and E4 regions, and the pIX showed a lesser homology with the corresponding proteins of other adenoviruses. The proteins V, VII, and IX are unusually long, and the protein VII lacks the consensus protease cleavage site. Genomic and cDNA sequence analysis has identified promoters, cap sites, intron-exon boundaries, polyadenylation signals, and polyadenylation sites in the viral genome.

Genetic organization and DNA sequence of early region 4 of bovine adenovirus type 3.

We have identified and sequenced 3614 nucleotides located at the extreme right-end of the bovine adenovirus type 3 (BAV3) genome from map units 89.5-100. Analysis of the sequence revealed an inverted terminal repeat (ITR) of 195 bp, and identified five open reading frames (ORFs) designated ORF1, ORF2, ORF3, ORF4 and ORF5. When compared with known E4 ORFs of other adenoviruses, ORFs 1, 2 and 4, which code for proteins of 143, 69 and 143 amino acids respectively, were found to be unique to BAV3. ORFs 3 and 5, which code for proteins of 268 and 219 amino acids respectively, showed partial homology to the E4 34 kDa protein of human adenovirus 2. Nucleotide sequence analysis also identified two potential TATA boxes upstream of ORF1 and a potential polyadenylation signal downstream of ORF5 suggesting that E4 transcripts may be 3' co-terminal.

Characterization of bovine adenovirus type 3 early region 2B.

We have determined the nucleotide sequence of a 6999 base pair region of bovine adenovirus-3 covering map units 9.0 to 29.17, which contained the adenovirus homologs of IVa2 protein and the DNA replication proteins, precursor of terminal protein and DNA polymerase proteins. Analysis of the sequence for cis-acting elements suggests that transcripts of DNA polymerase and precursor of terminal protein are 3' co-terminal. In addition, this region also contains major late promoter sequence. The sequence to the left of IVa2 contains the ORF of pIX with a potential TATA box immediately upstream and two polyadenylation consensus signals immediately downstream of the ORF.

Nucleotide sequence, genome organization, and transcription map of bovine adenovirus type 3.

The complete DNA sequence of bovine adenovirus type 3 is reported here. The size of the genome is 34,446 bp in length with a G+C content of 54%. All the genes of the early and late regions are present in the expected locations of the genome. However, the late-region genes are organized into seven families, instead of five as they are in human adenovirus type 2. The deduced amino acid sequences of open reading frames (ORFs) in the late regions and early region 2 (E2) and for IVa2 show higher degrees of homology, whereas the predicted amino acid sequences of ORFs in the E1, E3, and E4 regions and the pIX, fiber, and 33,000-molecular-weight nonstructural proteins show little or no homology with the corresponding proteins of other adenoviruses. In addition, the penton base protein lacks the integrin binding motif, RGD, but has an LDV motif instead of an MDV motif. Interestingly, as in other animal adenoviruses, the virus-associated RNA genes appear to be absent from their usual location. Sequence analysis of cDNA clones representing the early- and late-region genes identified splice acceptor and splice donor sites, polyadenylation signals and polyadenylation sites, and tripartite leader sequences.

Sequence and transcription map analysis of early region-1 of porcine adenovirus type-3.

The nucleotide sequence of a region of the genome of porcine adenovirus-3 (PAV-3) between map units 1 and 12.2 was determined. The sequenced region included four major open reading frames, and several transcription control elements. Homology studies, using the deduced amino acid sequences of the open reading frames, revealed genes coding for the E1A, E1B 202R, E1B 474R and pIX proteins. The region was characterized by Northern blot analysis and sequencing of cDNA clones. In PAV-3, the E1A region is located between 1.5 and 3.8 map units. Alternate splice donor sites are used to produce four different types of transcripts from the primary transcript of the E1A region. The E1A proteins of PAV-3 contain a consensus zinc finger motif, which was shown to be the principal transactivation region of human adenovirus-5 (HAV-5) E1A proteins. The PAV-3 E1A proteins also contain a retinoblastoma susceptibility protein (pRb) binding motif, which in HAVs interacts with cellular Rb protein to overcome the pRb mediated transcription repression. The E1B region in PAV-3 maps between 4.0 and 12.2 map units, and shares a polyadenylation signal and polyadenylation sites with the gene coding for pIX. A single major and a number of minor mRNA species are produced from the E1B region. The open reading frame (ORF) analysis of cDNA representing major mRNA produced from the E1B region showed two overlapping ORFs corresponding to 19K and 55K ORFs of HAV-2. In PAV-3, the gene coding for pIX is located between 9.9 and 12.2 map units and codes for a protein of 199 amino acids.

Restriction endonuclease analysis of equine herpesvirus-1 isolates recovered in Ontario, 1986-1992, from aborted, stillborn, and neonatal foals.

Ninety-two equine herpesvirus type 1 isolates were recovered from aborted, stillborn, or neonatal foals from Ontario, Canada, from 1986 to 1992. From this total, 32 strains were randomly chosen for further study. Four or 5 isolates from each winter were selected, each from a different premises, and characterized by restriction enzyme analysis using BamHI, KpnI, BglII, HindIII, and EcoRI. Additional isolates from 2 premises and from a zebra foal were also assessed. For the strains isolated in 1986 and 1989-1992, the DNA pattern of 18 strains was similar to that of type 1P (Kentucky D) for BamHI and KpnI. None of the 32 strains studied could be differentiated by HindIII or EcoRI. Using BglII, an inconsistent fragment pattern and distribution were observed. Of the 8 strains isolated in 1987 and 1988, 7 were assigned into the 1B prototype group. The geographic distribution of 17 type 1P and 12 1B isolates was random across southern Ontario. These findings suggest that both electropherotypes can be recovered from horses in Ontario. The patterns of the additional equine isolates from the same premises were identical. The zebra isolate was different from the prototype equine herpesvirus type 1 and type 4 patterns and from all other equine isolates.

Characterization of the early region 4 of porcine adenovirus type 3.

The nucleotide sequence of a 3028 bp DNA segment, located between map co-ordinates 100 and 92 in the genome of porcine adenovirus type 3 (PAV-3), was determined. The segment includes the entire early region 4 (E-4) and the right inverted terminal repeat sequences. There were two TATA boxes and one canonical polyadenylation signal on the 1 strand. Homology searches of the GenBank data base for the predicted amino acid sequences revealed that, of the eight open reading frames (ORFs) on the 1 strand, and four ORFs on the r strand, only ORF 8 on the 1 strand showed homology with the 34 kDa E-4 protein of human adenovirus types 2, 12 and 34. Northern blot analysis showed that transcription from the E-4 region of PAV-3 began 4 h after infection, peaked at 8 h and declined after 10 h, before DNA replication began 16 h after infection. The E-4 region of PAV-3 was further characterized by 5' and 3' end mapping of the transcription unit.

Porcine adenoviruses types 1, 2 and 3 have short and simple early E-3 regions.

The nucleotide sequence of the E-3 region genes, the hexon associated protein pVIII genes, and part of the fiber genes coding for the N-terminal tail regions, of porcine adenovirus (PAV) types 1 and 2 were determined. The sizes of the E-3 regions were found to be 1162 and 1222 bp, respectively. The five open reading frames (ORF) encoded within the sequenced regions of PAV types 1 and 2 shared a high degree of homology with the published sequences of the corresponding ORFs of PAV-3. The E-3 regions of PAV types 1, 2 and 3 were further characterized by Northern blot analysis and 5' and 3' end mapping of the transcripts by S1 nuclease analysis. The results of these experiments indicated that the E-3 regions in these three viruses are shorter and simpler in organization than the E-3 regions of human adenoviruses. A potential promoter for the E-3 regions of these PAVs was identified.