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Journal of Virology, May 2000, p. 4570-4578, Vol. 74, No. 10
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Expression of an Antigenic Adenovirus Epitope in a Group B Coxsackievirus

Katja Höfling, Steven Tracy,* Nora Chapman, Kyung-Soo Kim, and J. Smith Leser

Enterovirus Research Laboratory, Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska 68198-6495

Received 17 December 1999/Accepted 16 February 2000

Group B coxsackieviruses (CVB) cause human myocarditis, while human adenovirus 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 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. Progeny virus (CVB3-PL2-Ad2L1) was obtained following transfection of the construct into HeLa cells. 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. 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 cells. Cleavage of the CVB3-PL2-Ad2L1 polyprotein by 2Apro was slowed, accompanied by an accumulation of the fusion 1D-L1 loop protein. 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. 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. CVB3-PL2-Ad2L1 induced binding and neutralizing anti-Ad2 antibodies, in addition to antibodies against CVB3 in mice. 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. 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.

* Corresponding author. Mailing address: Enterovirus Research Laboratory, Department of Pathology and Microbiology, University of Nebraska Medical Center, 986495 Nebraska Medical Center, Omaha, NG 68198-6495. Phone: (402) 559-7747. Fax: (402) 559-4077. E-mail: stracy{at}unmc.edu.

Journal of Virology, May 2000, p. 4570-4578, Vol. 74, No. 10
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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