To present short protein sequences to the host immune system a foreign epitope has been expressed on the surface of the adenovirus virion as part of the hexon. As the trimeric hexon constitutes 240 out of the 252 capsomers of the virus, the foreign epitope is repeated 720 times on the virion surface. An eight amino acid sequence from the major antigenic site in the VP1 capsid protein of poliovirus type 3 was engineered into two regions of the adenovirus type 2 hexon. The two loop regions chosen to accommodate the foreign sequences are exposed on the surface of the virion, show sequence variation between serotypes and are the sites of interaction with neutralizing antibodies. Virus with substitutions in loop I had wild-type growth characteristics, whereas virus with substitutions in loop II grew poorly. Adenoviruses with poliovirus sequences in loop I were recognized and efficiently neutralized by antisera specific for the poliovirus sequence; an antiserum raised against the adenovirus with the poliovirus insert specifically recognized the VP1 capsid protein of poliovirus type 3. It is therefore feasible to alter the surface properties of the adenovirus virion and in doing so to manipulate the immune response to this virus. IntroductionHuman adenoviruses, of which 47 distinct serotypes have been identified (Hierholzer et al., 1988), replicate in the mucosal surfaces of respiratory, ocular and gastrointestinal tissues giving rise to a variety of acute infections. For over 30 years protection against infection by these viruses has been achieved by oral immunization which results in the generation of strong mucosal immunity. Live adenovirus types 4 and 7 (Ad4 and -7) in enteric coated capsules have been administered to military personnel to prevent acute respiratory disease with no indication of adverse reactions. Oral immunization with serotypes 1, 2 and 5 has also proved effective in clinical trials (reviewed in Ginsberg, 1984).Adenoviruses are non-enveloped and contain a linear double-stranded DNA genome of approximately 36 kb that is packaged within an icosahedral capsid. Hexon is the major structural component of the virus capsid, forming the 20 facets of the icosahedron; the vertices are composed of a complex of penton base and fibre (Ginsberg, 1984). Type-specific antigenic determinants of hexon have been demonstrated on the surface of the virion (Willcox & Mautner, 1976a, b) but the antigenic structure is complex with monoclonal antibodies (MAbs) defining at least 19 different epitopes on the surface of hexon (Russell et al., 1981 ;Adam et al., 1986). Sequence comparison of human adenovirus hexons and alignment with the three-dimensional structure of the adenovirus type 2 (Ad2) hexon (Roberts et al., 1986) indicates that although the base of the hexon is very highly conserved the surface loops are highly variable (Kinloch et al., 1984;Toogood & Hay, 1988;Toogood et al., 1989). The prediction that these variable regions could represent type-specific neutralizing determinants was confirmed by the demonstratio...

The adenovirus (Ad) hexon contains both group-and type-specific antigenic determinants. To identify the latter, peptides were synthesized corresponding to residues 281 to 292 from loop 1 and 441 to 455 from loop 2 of the Ad2 hexon. These sequences display typespecific variation and have been shown by X-ray crystallography to be present on the surface of the virion. Antisera raised against the peptides bound both peptide and the native hexon in ELISA, and blocked virus infectivity, as determined by immunofluorescence or neutralization assays. The loop 1 peptide was shown to inhibit binding of the corresponding antiserum to the native hexon in ELISA and to abolish its neutralizing activity. Neither the loop 1-nor loop 2-specific antiserum neutralized the infectivity of Ad4 or Ad40. Neutralization did not appear to result from aggregation of virus particles and thus their inability to attach to the cell, because virions treated with immune serum were internalized to the same extent as those treated with preimmune serum. Examination of the immune response elicited by Ad2 infection revealed that antibodies directed against the L 1 and L2 epitopes were also present in human serum. Thus, the variable regions exposed on the surface of the Ad2 hexon represent type-specific neutralizing antigenic determinants.

SUMMARYThe gene encoding the major capsid protein (hexon) of human adenovirus type 40 (Ad40) has been isolated and sequenced. Comparison of the predicted amino acid sequence of the Ad40 hexon with the corresponding polypeptide of the human enteric adenovirus, Ad41, reveals an overall identity of 88 ~. The majority of the changes in sequence are located in two areas, amino acids 131 to 287 and 390 to 425. Regions in the hexon protein that vary between Ad40 and Ad41 (subgroup F) were the same regions that varied between Ad2 and Ad5 (subgroup C) suggesting that these areas of the protein represent type-specific antigenic determinants. Other areas were conserved within members of a subgroup but varied between subgroups. Fitting of the Ad40 hexon sequence to the known three-dimensional structure of the Ad2 hexon demonstrates that the variable regions are located in the 11,12 and 14 loops that form the surface of the virion. Of major significance is the absence in Ad40 of the highly acidic region present in both Ad2 and Ad5. In Ad2 this region stretches down into the Dstrand of the fl-barrel forming the P1 domain. Molecular modelling indicates that the amino acids in Ad40 which correspond to the acidic region of Ad2 can also be accommodated in the eight-stranded fl-barrel, thereby maintaining the integrity of the barrel. Since the acidic region is also absent from the hexon of Ad41, the sequence of amino acids that replaces the acidic residues may be responsible for some of the distinctive biological properties of the subgroup F adenoviruses.

SUMMARYThe gene for the major capsid protein (hexon) of human adenovirus type 41 (Ad41) has been isolated and the complete DNA sequence determined. Comparison of the predicted amino acid sequence with hexons from human Ad2 and Ad5 and bovine adenovirus type 3 reveals regions of high homology at the N and C termini separated by a central region of low homology. Fitting of the Ad41 hexon sequence to the known three-dimensional structure of the Ad2 hexon demonstrates that both hexons have a common architecture. Regions of the hexon which in the trimer constitute the pseudohexagonal base are highly conserved, with the major amino acid changes concentrated in the domains forming the triangular towers which represent the surface of the capsid. Changes in the Ad41 towers therefore permit the virus to present a unique surface to the environment while conservation of residues in the base maintains the integrity of hexon-hexon contacts. A striking difference is the absence in the Ad41 sequence of 32 amino acids which are present in the Ad2 sequence. In Ad2 this region is highly charged and may be responsible for pH-induced conformational changes within the virus capsid. The DNA sequence in the region surrounding the Ad41 hexon gene was also determined and revealed an open reading frame which appeared to code for the homologue of the Ad2-coded endoprotease. Comparison of the predicted amino acid sequences of the Ad41 and Ad2 proteins revealed a high degree of homology suggesting that this protein may have an important role in the infectious cycle of the virus.

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