A Partial E3 Deletion 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; genetic stability; immune responses; vaccine.

Figure 1.

Partially E3-deleted adenovirus C (AdC) vectors are genetically stable and express the transgene product in transfected HEK-293 cells. (A) indicates the deletions. (B) shows the results of the restriction enzyme digest of the viral DNA isolated after an early (5th) or late (12th) passage. The molecular weight ladder (MW) is shown on the right of each gel. (C) shows vector vields for partially-(gp140) or E3-deleted (gag) AdC vectors. (D) shows Western blots for the AdC-Env vectors conducted with lysates of infected HEK-293 cells. Color images available online at www.liebertpub.com/hgtb

Figure 2.

AdC6-induced envelope protein (Env)–specific antibody responses. Groups of 5 mice were immunized with the vectors at the indicated doses and antibodies to the transgene product were measured in individual serially diluted sera harvested at baseline or 2, 4, or 8 weeks after immunization. Mice that received the 10¹⁰ virus particle (vp) dose were boosted with AdC7 vectors expressing the same transgene used for priming and sera were tested at 8 weeks after the boost (8wks pb). The graphs show adsorbance values as means with standard error of the mean (SEM) for serially diluted sera. The lines next to the legends show significant differences between the indicated groups by ANOVA. The statistical analyses were performed on area under the curve for each serum. ^–p > 0.05; *0.05 ≥ p > 0.01; **0.015 ≥ p > 0.001; ***0.001 ≥ p > 0.0001; ****p ≤ 0.00001.

Figure 3.

AdC7-induced Env-specific antibody responses. The graphs show results for sera from AdC7-immunized mice (as described for AdC6 in Fig. 2).

Figure 4.

Isotypes of vaccine-induced antibodies to Env. Stacked columns show mean adsorbance + SEM of Env-specific antibodies in sera of mice harvested at baseline, 8 weeks post priming (8 wks pp) or 8 weeks post boosting (8 wks pb). Lines with ^-/* indicate significant differences between the indicated groups, as in Fig 2. From left to right ^-/* are organized following the top-to-bottom list of the isotypes listed in the right lower corner of the graph.

Figure 5.

AdC-induced antibodies to the V2 loop. The graphs show adsorbance values as means with SEM for serially diluted sera from the same mice shown in Figs. 2 and 3. Sera harvested at baseline, at 8 weeks after priming, or at 8 weeks after the boost were tested. Lines with ^-/* show significant differences between the indicated groups again comparing area under the curve of individual sera.

Fig 6.

Affinity of AdC-induced antibodies. The graphs show the results of a NH₄SCN replacement assay conducted with a 1:200 dilution of sera from individual mice that had been immunized 8 weeks previously. Data are shown as percent adsorption in the presence of various concentrations of NH₄SCN compared with adsorption without NH₄SCN. The statistical analysis compared results at the same NH₄SCN concentrations between the two groups in each graph by multiple t-tests with Holms-Sidak correction. Symbols are as in Fig. 2.