Preventing spontaneous genetic rearrangements in the transgene cassettes of adenovirus vectors

Abstract

First-generation, E1/E3-deleted adenoviral vectors with diverse transgenes are produced routinely in laboratories worldwide for development of novel prophylactics and therapies for a variety of applications, including candidate vaccines against important infectious diseases, such as HIV/AIDS, tuberculosis, and malaria. Here, we show, for two different transgenes (both encoding malarial antigens) inserted at the E1 locus, that rare viruses containing a transgene-inactivating mutation exhibit a selective growth advantage during propagation in E1-complementing HEK293 cells, such that they rapidly become the major or sole species in the viral population. For one of these transgenes, we demonstrate that viral yield and cytopathic effect are enhanced by repression of transgene expression in the producer cell line, using the tetracycline repressor system. In addition to these transgene-inactivating mutations, one of which occurred during propagation of the pre-viral genomic clone in bacteria, and the other after viral reconstitution in HEK293 cells, we describe two other types of mutation, a small deletion and a gross rearranging duplication, in one of the transgenes studied. These were of uncertain origin, and the effects on transgene expression and viral growth were not fully characterized. We demonstrate that, together with minor protocol modifications, repression of transgene expression in HEK293 cells during viral propagation enables production of a genetically stable chimpanzee adenovirus vector expressing a malarial antigen which had previously been impossible to derive. These results have important implications for basic and pre-clinical studies using adenoviral vectors and for derivation of adenoviral vector products destined for large-scale amplification during biomanufacture.

Figure 1

Serial passage selected for ChAd63-CSN with the transgene interrupted by a bacterial transposon. A: Diagram of the expected structure (top) of the E1 region of ChAd63-CSN, showing viral inverted terminal repeat (ITR), “long” CMV promoter, CSN open reading frame (ORF), BGH polyadenylation (polyA) signal, and viral protein IX ORF (pIX); and (bottom) the IS150 bacterial insertion sequence disrupting the CSN ORF. Small white arrows in IS150 indicate the transposon ORFs. B: Discovery of the IS150 insertion by restriction mapping with BsrGI (B), NsiI (N), and PmeI (P) following 10 serial passages in comparison to starting virus and pre-viral plasmid. Aberrant bands are shown boxed. Marker band sizes are shown in base pairs (bp).

Figure 2

ChAd63-CSN isolate with a deletion at the CMV promoter splice acceptor site. A: Diagram of transgenic expression cassette of ChAd63-CSN showing “long” CMV promoter with its enhancer, exon, and intron; CSN open reading frame; and BGH polyadenylation (polyA) signal. Box indicates 66 bp deletion in the region of the splice acceptor site at the 5′ end of the intron, found in some viral isolates. B: Genomic restriction digest using BsrG1 of isolate A, with the deletion, and B, lacking the deletion, before and after 10 serial passages, in comparison to pre-viral plasmid. Arrowheads indicate the reduced size of the expected 1,870 bp band, which in the non-deleted samples co-migrates with the 1,947 bp band, resulting in a doublet in isolate A (passages 1 and 10), but not in the other samples. Figures on the left indicate expected sizes of viral digest bands and those on the right indicate marker sizes.

Figure 3

ChAd63-CSN isolates with transgene duplication and 5′ nonsense extension of CSN ORF. A: Diagram of the duplication observed in multiple viral isolates in the E1 region of the ChAd63-CSN genome, showing viral inverted terminal repeat (ITR); “long” CMV promoter; the variant duplicated CSN ORF (vCSN) with fusion to a duplicated CMV promoter at the 5′ end, resulting in a nonsense extension; the unmodified CSN ORF; the non-duplicated BGH polyadenylation (polyA) site; and the viral protein IX (pIX) ORF. Predicted restriction sites are indicated with sizes of aberrant bands shown. Compare to expected structure in Figure 1A. B: Genomic restriction digestions of ChAd63-CSN isolates B–D with the indicated enzymes, compared to pre-viral plasmid. Figures in the middle indicate marker band sizes; those on the left the predicted viral BsrGI fragments; and those on the right the predicted viral SnaBI fragments (predicted viral NsiI fragment sizes are not shown). White arrowheads indicate aberrant 744 bp (BsrGI) and 2,614 bp (NsiI and SnaBI) fragments resulting from the duplication shown in (A). Note that the absence of the 493 bp BsrGI and 890 bp SnaBI fragments from the plasmid is expected as a result of its circularity (unlike the linear viral genome).

Figure 4

Backbone of pre-viral plasmid inserted into adenoviral transgene. A: Diagram of the E1 region of ChAd63-Pfs230 (top) showing viral inverted terminal repeat (ITR), “long” CMV promoter, Pfs230 open reading frame (tPA-Pfs230 Region C), BGH polyA signal, and viral protein IX (pIX) open reading frame; and (bottom), a variant identified after rescue containing the pre-viral plasmid backbone (pUC) inserted into the transgene cassette with deletion of part of the promoter and most of the open reading frame, leaving only a fragment thereof (Frag). Small white arrow in pUC represents β-lactamase (AmpR) gene. B: Discovery of the variant shown in (A) by PCR analysis using primers in the CMV enhancer and the BGH polyA. NC, negative control; PP, pre-viral plasmid; V1, virus lysate after rescue; V2, virus after amplification and purification. Expected size = 3,428 bp; variant size = 2,777 bp, indicated by white arrowhead. C: Quantitative PCR assay for β-lactamase gene (AmpR) compared to CMV promoter, expressed as copy number per 5 μL for two limiting-dilution purified isolates (A and B) of ChAd63-CSN before (p0) or after (p10) 10 serial passages in HEK293 cells, compared to pre-viral plasmid (PP).

Figure 5

Repression of CSN expression improves growth of ChAd63-CSN. A: Diagrammatic representation of the E1 region of ChAd63-CSN genome showing modified transgenic expression cassettes incorporating TetO sequences immediately downstream of the TATA boxes of either the “long” (wild-type) version of the CMV promoter or the “short” version lacking any intron or splice-sites. Also shown are the viral inverted terminal repeat (ITR), the CSN open reading frame, the BGH polyadenylation signal (polyA), and the viral protein IX (pIX) open reading frame. B: Single-step growth of ChAd63-CSN with either “short” or “long” TetO-containing CMV promoter in HEK293 cells or HEK293 cells constitutively expressing the tetracycline repressor (TetR) at two multiplicities of infection (MoI). C: Phase contrast micrographs of cell monolayers showing onset of characteristic adenoviral cytopathic effect at 48 h in TetR-expressing HEK293 cells, but not in unmodified cells.

Figure 6

Immunogenicity, recombinant protein expression, and genetic stability of a redesigned ChAd63-CSN. A: Immunogenicity of CSN in Balb/c mice measured by ELIspot assay of splenocytes 14 days after vaccination with ChAd63-CSN containing either the “long” or “short” CMV promoter. Dots represent values for individual mice with line at mean and error bars showing standard error of the mean. SFC, spot-forming cells. B: Flow cytometry analysis of mean fluorescence intensity of HEK293 cells or TetR-HEK293 cells infected with ChAd63-CSN containing either the “long” or “short” CMV promoter and labeled with anti-CS monoclonal antibody. Error bars indicate standard error of the mean of three replicates. C: Quantification of CSN protein by capture ELISA in lysates of A549 cells transduced with ChAd63-CSN containing either the “long” or “short” CMV promoter. Error bars indicate standard error of the mean of two replicates. D: Genomic restriction digestions using indicated enzymes of two isolates of ChAd63-CSN (“short”) before and after 10 passages in either HEK293 or TetR-HEK293 cells, compared to pre-viral plasmid.