Replication-defective vector based on a chimpanzee adenovirus

Abstract

An adenovirus previously isolated from a mesenteric lymph node from a chimpanzee was fully sequenced and found to be similar in overall structure to human adenoviruses. The genome of this virus, called C68, is 36,521 bp in length and is most similar to subgroup E of human adenovirus, with 90% identity in most adenovirus type 4 open reading frames that have been sequenced. Substantial differences in the hexon hypervariable regions were noted between C68 and other known adenoviruses, including adenovirus type 4. Neutralizing antibodies to C68 were highly prevalent in sera from a population of chimpanzees, while sera from humans and rhesus monkeys failed to neutralize C68. Furthermore, infection with C68 was not neutralized from sera of mice immunized with human adenovirus serotypes 2, 4, 5, 7, and 12. A replication-defective version of C68 was created by replacing the E1a and E1b genes with a minigene cassette; this vector was efficiently transcomplemented by the E1 region of human adenovirus type 5. C68 vector transduced a number of human and murine cell lines. This nonhuman adenoviral vector is sufficiently similar to human serotypes to allow growth in 293 cells and transduction of cells expressing the coxsackievirus and adenovirus receptor. As it is dissimilar in regions such as the hexon hypervariable domains, C68 vector avoids significant cross-neutralization by sera directed against human serotypes.

Figures

FIG. 1

Genetic organization of the C68 genome. (A) The genome of the C68 chimpanzee adenovirus is schematically represented by the box at the top. The inverted terminal repeats are shaded black, and the early regions are shaded gray. The arrowheads above the box indicate the direction of expression of the early genes. The line below the box represents the division of the genome into 100 m.u. The arrows below the line represent the five late gene regions and the proteins encoded in each region. The numbers below the box or arrows indicate the start (promoter or initiation codon) and end (canonical polyadenylation signal) for each region. ∗ represents the E2A late promoter. (B) PstI clones. (C) BamHI clones. (D) HindIII clones. The unshaded regions indicate that a fragment was cloned into a plasmid vector, as listed in Table 1, while the shaded regions indicate that the restriction fragment was not cloned. For each section, the fragment name, alphabetical with A being the largest fragment, and the fragment size are listed above the box, and the fragment end points are listed below the box.

FIG. 2

Pairwise genomic sequence comparisons between C68 and selected adenoviruses from various subgroups. The genome of each human serotype was aligned to C68, and percent identity plots (pips) were generated with PipMaker. Since only partial sequence information was available for adenovirus types 4 (Ad4) and 7, minigenomes were created and used in the analysis. Pips are arranged from top to bottom in order of decreasing identity. Regions of interest are highlighted in gray. Only segments showing greater than 50% identity to C68 are shown in the plots. In the case of adenovirus types 4 and 7, gaps of less than 50% identity represent regions for which an alignment was not generated due to insufficient input data. Arrows indicate the orientation, size, and location of genes of interest. Black boxes designate coding sequences, and numbers indicate interruptions in those sequences. Short gray/white boxes illustrate CpG island composition (CpG/GpC ratio), with white indicating 0.60 to 0.75 and gray greater than 0.75. The following GenBank documents were used in the alignment: NC001405 (adenovirus type 2), NC001406 (adenovirus type 5), NC002067 (adenovirus type 17), NC001460 (adenovirus type 12), and NC001464 (adenovirus type 40).

FIG. 3

Schematic description of a generic vector construction process for replication-defective C68 viruses. Step A, C68 fragment spanning bp 4012 to 6099 was removed from pC68-CMV-AP byAsp718 and AgeI endonuclease treatments and replaced by an AgeI/BsiWI fragment. This fragment, containing bp 4012 to 11710 of C68 DNA, was isolated from pBS-C68 Bam-B. Step B, pC68-CMV-AP-MU32 was restricted withEco47III and NruI endonucleases to delete a 1.72-kb fragment from C68 DNA and self-ligated. Step C, a part of the CMV promoter, alkaline phosphatase cDNA, and SV40 poly(A) addition signal was removed from pC68-CMV-AP-MU26 by SnaBI andPsiI digestions and substituted with aSnaBI-EcoRI fragment. This fragment, consisting of a part of the CMV promoter, prokaryotic GFP expression cassette, and bovine growth hormone poly(A) addition signal was obtained from pShut-GFPmut3-1. Step D, the prokaryotic GFP expression cassette was released from pC68-CMV-PkGFP-BGHPa-MU26 afterNheI and PmeI digestions and replaced with the cDNA of the gene of interest.

FIG. 4

Neutralizing antibodies against adenovirus type 5 and C68. Sera from 50 normal human subjects, 52 rhesus monkeys, and 20 chimpanzees were analyzed for neutralizing antibody against human adenovirus type 5 (A) or C68 (B). The percentage of total serum samples that titrated at the indicated dilutions is presented.

FIG. 5

Analysis of sera from immunized mice for neutralization of human adenovirus type 5 and C68. Groups of C3H/He mice were immunized subcutaneously with 4 × 1010 replication-competent adenovirus particles per mouse. Mice were bled 18 days later. Sera as well as a control serum from naive mice (NMS) were tested for virus-neutralizing activity (VNA) against adenovirus type 5 (Ad5)-GFP (striped bars) and C68-GFP (solid bars) virus.

FIG. 6

Multiple sequence alignment of hexon proteins. The deduced amino acid sequences of highly similar human adenovirus hexons were compared with the chimpanzee adenovirus using Clustal X. Serotypes and subgroups are indicated on the left margin, followed by the residue number. The numbering refers to the amino acid position with respect to the start of translation. Amino acids are shaded with respect to C68 to highlight sequence similarities (gray) and identities (black). The seven hypervariable regions within loop domains DE1 and FG1 are labeled along the bottom and correspond to the following adenovirus type 2 sequences in the alignment: hypervariable region 1, 137 to 188; hypervariable region 2, 194 to 204; hypervariable region 3, 222 to 229; hypervariable region 4, 258 to 271; hypervariable region 5, 278 to 294; hypervariable region 6, 316 to 327; and hypervariable region 7, 433 to 465. The GenBank accession numbers for the sequences shown are as follows: AAD03657 (adenovirus type 4), S37216 (adenovirus type 16), S39298 (adenovirus type 3), AAD03663 (adenovirus type 7), and NP040525 (adenovirus type 2).

FIG. 7

Predicted structure of C68 hexon. Ribbon representation of the C68 hexon homology model structure. (A) Trimer viewed from the top (exterior surface of virion); (B) trimer viewed from the side; (C) monomer. The structure is colored to indicate the sequence diversity between C68 and human adenovirus type 4. The colors range from blue (conserved) to yellow (conservative) to red (nonconservative). The four upper loops (DE1, FG1, DE2, and FG2) are labeled. DE1 contains hypervariable regions 1 to 6, and FG1 contains hypervariable region 7.