Identification of adenovirus (ad) penton base neutralizing epitopes by use of sera from patients who had received conditionally replicative ad (addl1520) for treatment of liver tumors

Abstract

Sera from 17 patients with primary and secondary liver tumors who had been administered oncolytic adenovirus (Ad) mutant Addl1520 were analyzed for anti-Ad neutralization titers and antibodies to the Ad major capsid proteins hexon, penton base (Pb), and fiber. The antibodies recognized mainly conformational epitopes in hexon and both linear and conformational epitopes in Pb and fiber. Pb-specific antibodies were isolated from serum samples that had been obtained prior to and during the course of the treatment of four of these patients. We found that the Pb antibodies had a significant contribution toward anti-Ad neutralization, and this mainly occurred at the step of virus internalization. The Pb antigenic epitopes were determined by phage biopanning and were mapped to 10 discrete regions, which made up three major immunodominant domains within residues 51 to 120, 193 to 230, and 311 to 408, respectively. One of these domains (residues 311 to 408) overlapped the highly conserved, integrin-binding RGD (Arg-Gly-Asp) motif. The contribution of antibodies directed to RGD and other epitopes in Ad neutralization activity was determined indirectly by using a phage-mediated depletion assay. Our results suggested that circulating RGD antibodies were not prevalent and were poorly neutralizing and that other peptide motifs within residues 51 to 60, 216 to 226, and 311 to 408 in Pb sequence represented major target sites for neutralizing antibodies.

FIG. 1.

Western blot analysis of sera from patients 1, 2, 3 and 4 taken before (day 0 [D0]) or after (day 10 [D10]) Addl1520 administration. Aliquots of serum dilutions were reacted with membrane strips transferred with recombinant Ad Pb protein electrophoresed under native conditions (a) or a mixture of SDS-denatured hexon, Pb, and fiber proteins separated by conventional SDS-PAGE (b and c). Control samples (lane 9 in panel a; lanes 9 to 11 in panel b; lanes 5 and 6 in panel c) consisted of strips that were reacted with rabbit anti-fiber (αFi), anti-Pb (αPb), or anti-whole Ad virion (αAd) antibodies, respectively.

FIG. 2.

Virus NA activity of Pb antibodies from human sera. (a) Global effect of whole sera; (b) global effect of Pb-specific antibodies; (c) effect of Pb-specific antibodies at the endocytotic step. In panel a, Ad5Luc3 recombinant was preincubated without (control, no antibody [con]) or with total serum samples from patients 1 (P1), 2 (P2), 3 (P3), or 4 (P4) (using the D0 sera of patients 1 and 2 and the D10 sera of patients 3 and 4) and then incubated with HeLa cells. In panel b, Ad5Luc3 was preincubated with or without isolated Pb antibodies. In panel c, isolated Pb antibodies were added to virus-cell monolayers after preattachment of Ad5Luc3 to HeLa cells at 4°C for 30 min. In the three sets of experiments (i.e., panels a to c), virus infection was allowed to proceed for 18 h at 37°C, and the cells were processed for luciferase assays. The NA effect was indirectly assayed by the level of luciferase activity expressed as arbitrary units and then normalized to the percentage of the control samples.

FIG. 3.

Epitope mapping in Ad Pb protein. The Pb sequence is represented linearly. Under the Pb line are shown the positions of the epitopes defined in Table 2. The patient sera reacting with the different epitopes are indicated in parentheses. Above the Pb line are shown the major IDRs in humans, as defined in the present study, and in mice, after immunization with recombinant Pb (11). The probability of immunogenicity (as evaluated by the antigenic index of Jameson-Wolf) is presented at the top of the figure.

FIG. 4.

Phage-mediated depletion of Pb epitope-specific antibodies. Phages carrying the Pb epitopes indicated at the top of each panel were amplified and incubated with D10 serum samples from patients 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 14, and 16, as well as samples 18, 19, 20, and 21 from healthy donors. After removal of the phage-antibody complexes by centrifugation, the serum supernatants were assayed for their effect on Ad-mediated gene transfer by using the replication-competent Ad5Luc3 vector. Luciferase expression was compared in cells infected with Ad5Luc3 treated with depleted or nondepleted sera. Theoretically, values greater than 1.0 for the ratio of luciferase levels between depleted and nondepleted serum samples indicated a decrease in NA activity after phage depletion but, once the experimental errors are taken into account, only values of >1.3 were considered significant.