Virus-specific neutralizing antibodies present an obstacle to the effective use of adenovirus vectors for gene therapy and vaccination. The specific sites recognized by neutralizing antibodies have not been identified for any adenovirus, but they have been proposed to reside within the hexon, in small regions of the molecule that are exposed on the capsid surface and possess sequences that vary among serotypes. We have mapped the epitopes recognized by a panel of seven hexon-specific monoclonal antibodies that neutralize the chimpanzee adenovirus 68 (AdC68). Surface plasmon resonance experiments revealed that the antibodies compete for a single hexon binding site, and experiments with synthetic peptides indicated that this site resides within just one small surface loop. Mutations within this loop (but not in other surface loops) permitted virus to escape neutralization by all seven monoclonal antibodies and to resist neutralization by polyclonal antisera obtained from animals immunized against AdC68. These results indicate that a single small surface loop defines a major neutralization site for AdC68 hexon.Modified adenoviruses have been widely used as vehicles for gene delivery and as vaccine vectors. Most adenovirus vectors in use have been derived from the human serotype 5 (Ad5). As almost all human adults have been exposed to Ad5, they possess neutralizing antibodies to Ad5 that limit the efficiency of the virus as a delivery vector (7,33,42). Approaches to circumvent the problem of preexisting immunity include chemical modification of Ad5 surface proteins to mask the neutralizing epitopes (4,19,29) and replacement of the immunogenic capsid proteins with those of other serotypes (21,23,32,41,44). Many investigators are also exploring the use of rare human serotypes (such as human Ad48) or nonhuman adenoviruses (including those derived from dogs, fowl, and nonhuman primates) to which humans are not usually immune (2, 6, 13, 16-18, 22, 30).An alternative approach is to identify the specific sites on adenovirus that are recognized by neutralizing antibodies and then modify those sites to generate mutants capable of escaping neutralization. One nonhuman serotype that has been proposed as an alternative vector for vaccination is the chimpanzee adenovirus 68 (AdC68) (42). AdC68 is not neutralized by most human adult sera and elicits a strong transgene productspecific immune response in animals already immune to Ad5 (7,33,42). However, because one immunization with an AdC68 vector will induce serotype-specific immunity, multipledose immunization regimens may require the availability of additional vectors. Production of antigenically modified vectors would be facilitated if the epitopes recognized by the neutralizing antibodies were well characterized.The adenovirus capsid is an icosahedron with long fibers projecting from the vertices. Twelve copies of the trimeric major capsid protein, hexon, form each of the 20 triangular facets of the icosahedron; trimeric fibers are inserted into the pentameric penton bases at...

A major obstacle to the use of adenovirus vectors derived from common human serotypes, such as human adenovirus 5 (AdHu5), is the high prevalence of virus-neutralizing antibodies in the human population. We previously constructed a variant of chimpanzee adenovirus 68 (AdC68) that maintained the fundamental properties of the carrier but was serologically distinct from AdC68 and resisted neutralization by AdC68 antibodies. In the present study, we tested whether this modified vector, termed AdCDQ, could induce transgene product-specific CD8 ؉ T cells in mice with preexisting neutralizing antibody to wild-type AdC68. Contrary to our expectation, the data show conclusively that antibodies that fail to neutralize the AdCDQ mutant vector in vitro nevertheless impair the vector's capacity to transduce cells and to stimulate a transgene product-specific CD8 ؉ T-cell response in vivo. The results thus suggest that in vitro neutralization assays may not reliably predict the effects of virus-specific antibodies on adenovirus vectors in vivo.