(a) Amplimers were separated by agarose gel electrophoresis. Viral RNA was isolated from virus stocks at each passage, and the presence of the inserted Ad2 sequence was analyzed by PCR using primers flanking the insertion site in the CVB3 genome. pCVB3-PL2-Ad2L1 was transfected into HeLa cells, and the resultant progeny virus (CVB3-PL2-Ad2L1, pass 1) was subsequently serially passaged in HeLa cell cultures (passes 2 to 10). PCR and sequence analysis of CVB3-PL2-Ad2L1. The data demonstrate that a CVB 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. CVB3-PL2-Ad2L1 was used to challenge mice previously inoculated with CVB3/0 and with preexisting anti-CVB3 neutralizing-antibody titers anti-Ad2 neutralizing and binding antibodies were induced in these mice at higher levels than in mice without anti-CVB3 immunity. CVB3-PL2-Ad2L1 induced binding and neutralizing anti-Ad2 antibodies, in addition to antibodies against CVB3 in mice. Mice inoculated with CVB3-PL2-Ad2L1 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. Reverse transcription-PCR sequence analysis of CVB3-PL2-Ad2L1 RNA demonstrated that the Ad2 hexon polypeptide coding sequence was maintained in the chimeric viral genome through at least 10 passages in HeLa cells. Cleavage of the CVB3-PL2-Ad2L1 polyprotein by 2Apro was slowed, accompanied by an accumulation of the fusion 1D-L1 loop protein. Viral proteins were detected earlier and in approximately fourfold greater amounts in CVB3-PL2-Ad2L1-infected HeLa cells than in CVB3/0-infected cells. Western blot analyses of the CVB3 capsid protein 1D in CVB3-PL2-Ad2L1-infected HeLa cells demonstrated production of the expected capsid protein. Replication of CVB3-PL2-Ad2L1 in diverse cell cultures demonstrated that the yield of the chimeric virus was between 0.5 to 2 log units less than the parental strain. Progeny virus (CVB3-PL2-Ad2L1) was obtained following transfection of the construct into HeLa cells.
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To evaluate the feasibility of a multivalent vaccine strain against the CVB and Ad2, we cloned the sequence encoding the Ad2 hexon L1 loop, flanked by dissimilar 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. The L1 loop of the Ad2 hexon protein has been demonstrated to be antigenic in rabbits.
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Group B coxsackieviruses (CVB) cause human myocarditis, while human adenovirus type 2 (Ad2) is implicated as an agent of this disease.