A PartialE3Deletion in Replication-Defective Adenoviral Vectors Allows for Stable Expression of Potentially Toxic Transgene Products

Abstract

Adenovirus (Ad) is used extensively for construction of viral vectors, most commonly with deletion in its E1 and/or E3 genomic regions. Previously, our attempts to insert envelope proteins (Env) of HIV-1 into such vectors based on chimpanzee-derived Ad (AdC) viruses were thwarted. Here, we describe that genetic instability of an E1-and E3-deleted AdC vector of serotype C6 expressing Env of HIV-1 can be overcome by reinsertion of E3 sequences with anti-apoptotic activities. This partial E3 deletion presumably delays premature death of HEK-293 packaging cell lines due to Env-induced cell apoptosis. The same partial E3 deletion also allows for the generation of stable glycoprotein 140 (gp140)-and gp160-expressing Ad vectors based on AdC7, a distinct AdC serotype. Env-expressing AdC vectors containing the partial E3 deletion are genetically stable upon serial cell culture passaging, produce yields comparable to those of other AdC vectors, and induce transgene product-specific antibody responses in mice. A partial E3 deletion thereby allows expansion of the repertoire of transgenes that can be expressed by Ad vectors.Keywords: HIV-1, vaccine, genetic stability, immune responses INTRODUCTION REPLICATION-DEFECTIVE ADENOVIRUS (Ad) vectors efficiently deliver transgenes to multiple cell types. 1,2 They trigger a potent inflammatory response, 3,4 which initiates adaptive immune responses to the transgene product and the antigens of the Ad vector. 5 This has led to the development of different Ad serotypes as vaccine carriers for a plethora of antigens derived from viruses, 6-10 bacteria, 11,12 parasites, 13,14 or tumor cells, [15][16][17] which have consistently induced potent and sustained B and T cell responses to the transgene products.In most Ad vaccine vectors, the E1 and E3 domains are deleted from the genome. The E1 deletion renders Ad vectors replication defective and reduces synthesis of other Ad antigens. E3 encodes a number of polypeptides that have anti-apoptotic activity or allow the virus to escape immune surveillance. 18,19 The E3 domain, a region that is nonessential for viral replication, is generally deleted to allow for the insertion of lengthy transgene expression cassettes without exceeding the genome size of wild-type virus, as this could potentially result in genetic instability of Ad vectors. 20 Ad vectors have been explored extensively as vaccine carriers for antigens of human immunodeficiency virus (HIV)-1, 21-25 the causative agent of the acquired immunodeficiency syndrome (AIDS). While initial efforts focused on expression of internal antigens for the induction of CD8 + T cell responses against HIV-1, efforts have since shifted toward expression of the envelope protein (Env). The heavily glycosylated and highly variable Env of HIV-1 forms trimers composed of glycoprotein (gp) 120 and gp41 on the surface of the virion. These complexes allow HIV-1 to attach to its receptors and coreceptors and are thereby essential for CD4 + cell infection by HIV-1. Env is not only the sole target of HIV-1-specif...

Generation and Characterization of a Replication-Competent Human Adenovirus Type 55 Encoding EGFP

“…The E3 gene is a non-essential region for adenovirus replication in cell culture. The deletion of the E3 region does not affect viral replication and proliferation, while the insertion of the transgene into the E3 14.7K region produces a high level of tumor-selective transgene expression [ 40 ]. The E3 region is an early expressed protein, and foreign genes inserted into the E3 region are more likely to be highly expressed [ 41 ].…”

Porcine Adenovirus Type 3 E3 Encodes a Structural Protein Essential for Capsid Stability and Production of Infectious Progeny Virions

“…The isolation of a viable mutant PAV13.7K 3A (not expressing 13.7K) using the VIDO AS2 cell line (providing 13.7K protein in trans) confirms that 13.7K is essential for efficient replication of PAdV-3. However, the HAdV type 5 E3-encoded 12.5K protein showing limited homology (40%) to PAdV-3 13.7K does not appear to be essential for viral replication (18). Interestingly, homologs of PAdV-3 ORF2 in PAdV-1 and PAdV-2 show amino acid identities of 97.4% and 94%, respectively, with PAdV-3 ORF2 (19), suggesting that PAdVs may have a unique feature of encoding an E3 protein, which appears to be essential for virus replication.…”

“…Despite divergence in the size and number of genes carried by the adenovirus E3 region (10,(15)(16)(17)(18), a positional homolog of the E3 region appears to be conserved in members of Mastadenovirus (10-16), suggesting an important role for E3 in the virus life cycle (17,(31)(32)(33). The proteins encoded by the E3 region not only help in immune evasion by adenovirus (32), including modulation of leukocyte functions (31), but are also involved in inducing lytic infection of lymphocytes (33) and helping in the stable expression of toxic transgene products in adenovirus vectors (18). In spite of the preservation of the E3 region and its important role in the adenovirus life cycle, none of the proteins encoded by adenovirus E3 regions appear to be a component of the progeny virion or required for adenovirus replication (18).…”

“…The proteins encoded by the E3 region not only help in immune evasion by adenovirus (32), including modulation of leukocyte functions (31), but are also involved in inducing lytic infection of lymphocytes (33) and helping in the stable expression of toxic transgene products in adenovirus vectors (18). In spite of the preservation of the E3 region and its important role in the adenovirus life cycle, none of the proteins encoded by adenovirus E3 regions appear to be a component of the progeny virion or required for adenovirus replication (18). In the present study, we demonstrate that 13.7K protein encoded by the PAdV-3 E3 region is incorporated in the mature and immature virions and appears to be essential for the replication of PAdV-3 and stability of virion capsid.…”

“…The adenovirus E3 region is complex and produces several overlapping mRNAs, encoding different proteins (17). Although the E3 transcription unit is nonessential for replication of adenovirus (18) and can be used for insertion of transgenes (6,7,17,18), the efficient expression of some transgenes requires the presence of some E3 genes (18).…”