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

Abstract

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.

Recombinant Bovine Adenovirus Type 3 Expressing Bovine Viral Diarrhea Virus Glycoprotein E2 Induces an Immune Response in Cotton Rats

“…One reason could be the presence of internal cryptic splice sites in E2ori, which could result in the synthesis of inappropriate mRNAs in the nucleus. However, insertion of an intron upstream of E2ori did not alter the expression of the protein (data not shown), although a similar approach was successful in obtaining desirable expression of bovine corona virus E3 glycoprotein in the E3 region of BAV-3 (Reddy et al, 2000). Alternatively, it is possible that the transcripts are unstable in the nucleus (Kühnle et al, 1998).…”

Xenogenic Adenoviral Vectors

“…The addition of exogenous control elements comprising an intron and the HCMV or SV40 promoter increased the level of expression but altered the kinetics. The recombinant virus also replicated less efficiently than wild-type BAdV3 [142].…”

Bovine adenovirus-3 as a vaccine delivery vehicle

“…The E3 region of BAdV-3 is 1.591 kb. Since E3 region is non essential for replication of adenoviruses [46,55], initial attempts resulted in isolating a viable recombinant BAdV-3 containing deletion of 1.245 kb of E3 region [46]. Although potential insertion capacity of E3 deleted vector is 3 kb [46], the insertion of 2.8 kb foreign DNA [56] has been successful in generating viable replication-competent recombinant BAdV-3.…”

“…Moreover, addition of exogenous consensus sequence for polyadenylation of foreign ORF affects the replication efficiency of recombinant BAdV-3 (Lobanov and Tikoo, unpublished data). Interestingly, efficient expression of a RNA virus required addition of an intron and consensus sequence for polyadenylation upstream and downstream, respectively, of the gene [55]. Since E3 deleted recombinant BAdV-3 are replication competent (Fig.…”

“…Earlier, successful expression of a reporter ORF inserted in partially deleted E3 region of BAdV-3 [45] suggested the feasibility of utilizing E3 region for expression of foreign genes. Thus, subsequent expression of different forms of (BHV)-1 vaccine antigen gD ORF [46], (BVDV) E2 ORF [57] or (BCV) virus HE ORF [55] inserted individually in fully deleted E3 region of BAdV-3 demonstrated the potential of developing BAdV-3 based recombinant vaccines for cattle. These ORFs were selected as earlier reports have suggested the potential of bovine herpesvirus (BHV)-1 glycoprotein gD [59], bovine viral diarrhea virus (BVDV) E2 glycoprotein [60] 40 78 87 Late (L) Region corona virus (BCV) hemagglutinin ORF [61] to induce protective immune responses in animals and thus act as vaccine antigen.…”