Expression of an antigenic adenovirus epitope in a group B coxsackievirus

Group B coxsackieviruses (CVB) cause human myocarditis, while human adenovi rus type 2 (Ad2) is implicated as an agent of this disease. The L1 loop of the Ad2 hexon protein has been demonstrated to be antigenic in rabbits. To evaluate the feasibility of a multivalent vaccine strain against the CVB an d Ad2, we cloned the sequence encoding the Ad2 hexon L1 loop, flanked by di ssimilar sequences encoding the protease 2A (2Apro) recognition sites, into the genome of an attenuated strain of CVB type 3 (CVB3/0) at the junction of 2Apro and the capsid protein 1D. Progeny virus (CVB3-PL2-Ad2L1) was obta ined following transfection of the construct into HeLa cells. Replication o f CVB3-PL2-Ad2L1 in diverse cell cultlures demonstrated that the yield of t he chimeric virus was between 0.5 to 2 log units less than the parental str ain, Western blot analyses of the CVB3 capsid protein 1D in CVB3-PL2-Ad2L1- infected HeLa cells demonstrated production of the expected capsid protein. Viral proteins were detected earlier and in approximately fourfold greater amounts in CVB3-PL2-Ad2L1-infected HeLa cells than in CVB3/0-infected cell s. Cleavage of the CFTB3-PL2-Ad2L1 polyprotein by 2Apro was slowed, accompa nied by an accumulation of the fusion 1D-L1 loop protein. Reverse transcrip tion-PCR sequence analysis of CVB3-PL2-Ad2L1 RNA demonstrated that the Ad2 hexon polypeptide coding sequence was maintained in the chimeric viral geno me through at least 10 passages in HeLa cells. Mice inoculated with CVB3-PL 2-Bd2L1 demonstrated a brief viremia with no replication detectable in the heart but prolonged replication of virus in the pancreas in the absence of pathologic changes in either organ. CVB3-PL2-Ad2L1 induced binding and neut ralizing anti-Ad2 antibodies, in addition to antibodies against CVB3 in mic e, CVB3-PL2-Ad2L1 was used to challenge mice previously inoculated with CVB 3/0 and with preexisting anti-CVB3 neutralizing-antibody titers; anti-Ad2 n eutralizing and binding antibodies were induced in these mice at higher lev els than in mice without anti-CVB3 immunity. The data demonstrate that a CV B vector can stably express an antigenic polypeptide of Ad2 from within the CVB open reading frame that results in the induction of protective immune responses against both viruses.