Identification of Adenovirus (Ad) Penton Base Neutralizing Epitopes by Use of Sera from Patients Who Had Received Conditionally Replicative Ad (Ad dl 1520) 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.

Neutralizing Antibodies to Adenovirus Serotype 5 Vaccine Vectors Are Directed Primarily against the Adenovirus Hexon Protein

“…Previous studies have also shown that chimeric rAd5 vectors containing the Ad2, Ad6, or Ad12 hexon genes were able to evade a substantial fraction of anti-Ad5 immunity in rodents (20 -22), although these studies did not differentiate between the contributions of hexon-specific humoral and cellular immunity. In contrast with these reports, a recent clinical study suggested that fiber-and penton-specific Abs were in fact substantially higher titer than hexon-specific Abs in humans using Western blot analyses (23). Another clinical study similarly reported that Ad5-specific Abs recognized primarily fiber and penton determinants and that these Abs exhibited synergistic neutralizing activity (24).…”

“…Moreover, studies in rodents using hexon-chimeric viruses have suggested that hexon-specific immune responses also contribute substantially to anti-Ad5 immunity in vivo (20 -22). In contrast, recent clinical studies have reported that fiber-and penton-specific Abs were more frequent and higher titer than hexon-specific Abs in humans (23) and that fiber-and penton-specific Abs exerted synergistic neutralizing activity (24). It is clear from these studies that Ad5-specific Abs against multiple capsid components are generated.…”

“…In contrast, fiber-and penton-specific NAbs have substantially slower kinetics of neutralizing free virus and are unable to neutralize viral particles following attachment to cells (19). Fiber-specific NAbs are thought to function by inducing aggregation of viral particles before cellular attachment, and pentonspecific NAbs may inhibit viral uncoating (19,23). These kinetic and mechanistic differences may contribute to the differences in efficiency among hexon-, fiber-, and penton-specific NAbs in suppressing rAd5 vaccine immunogenicity in vivo.…”

“…We speculate that the differences between our results and these prior clinical studies may reflect the efficiency with which hexon-specific Abs neutralize viral particles. As a result, Western blots using purified proteins (23,24) may underestimate the functional capacity of hexon-specific Abs to neutralize virus.…”

Osteosarcoma cells as carriers to allow antitumor activity of canine oncolytic adenovirus in the presence of neutralizing antibodies

“…27 However, viral protein expression on the cell surface does not possess the same importance for adenoviruses as for other OVs as Nabs recognize viral proteins not expressed on the carrier-cell surface. 28 We performed short infection times as a measure of safeguard. In our studies, the loading time was set to 4 h, as this is the minimum amount of time necessary to infect a substantial proportion of carrier cells.…”