Characterization of the neutralizing epitopes within HVR5 of HAdv-7.

<p>(A) Immunoblotting analysis indicated that the four HAdv-7-neutralizing MAbs recognized hexon in trimeric form (T) at room temperature (RT), but not the hexon monomers (M) at room temperature or heated at 95°C for 5 min, in the presence of SDS. HEp-2-cell-cultured rAdMHE3 virions were harvested and stored at room temperature or heated at 95°C for 5 min in the presence of SDS. The samples were then separated by SDS-PAGE and tranferred onto nitrocellulose membranes. The membranes were then cut into individual strips and incubated with individual MAb or antiserum. After the blot being developed, the strips were spliced and their signals were exposed to two pieces of X films. The MAb 3D7, which bound to the recombinant HAdv-3 hexon protein, and the anti-rAdMHE3 serum were used as the positive controls. Lane 1, 1C7; lane 2, 3G5; lane 3, 1D9; lane 4, 6F3; lane 5, 3D7; lane 6, anti-rAdMHE3. *The rAdMHE3 virus suspensions were stored at −20°C for several months, so there may be degradation for some hexon, which may explain the minor bands of less than 120 kDa. (B) HAdv-7 virions, recombinant truncated hexon fragments of HAdv-3 (A3H) or HAdv-7 (A7nH), and BSA-conjugated synthetic HAdv-7 HVR5 peptide (BSA-A7R5) were reacted with the MAbs or different antisera in indirect ELISAs. The MAbs did not react with BSA-A7R5, A3H, or A7nH. Both anti-KLHA7R5 and anti-GSTA7R5 bound to HAdv-7 virions weakly. In addition, anti-rAd3egf/H7 bound to BSA-A7R5 weakly as well. Each experiment was repeated independently at least three times, and the mean values and standard deviations (indicated by the error bars) are shown.