DNA vaccines prime CD8+ T cell responses to epitopes of viral antigens produced from overlapping reading frames of a single coding sequence.

A hepatitis B virus (HBV)-derived sequence that encodes the 832-residue polymerase (Pol) protein of HBV in the primary open reading frame (ORF), and the three (large, middle and small) hepatitis B surface antigen (HBsAg) variants in an alternative ORF was used. This sequence was cloned into expression vectors in which Pol was expressed under heterologous (HCMV, SV40 or metallothionin) promoter control. Some Pol-encoding vectors coexpressed Pol as well as readily detectable amounts of HBsAg. Efficient HBsAg expression depended on endogenous HBV promoter sequences but was apparently also facilitated by heterologous promoter sequences located upstream of the HBV Pol sequence. DNA immunization of mice efficiently coprimed CD8(+) T cell responses to epitopes of Pol and HBsAg. Over expression of Pol (using an hsp73-facilitated expression system) did not correlate with the immunogenicity of the K(d)/Pol(140-148) epitope. Immunodominant L(d)-restricted CD8(+) T cell responses to HBsAg down-modulated priming of CD8(+) T cell responses to other HBsAg epitopes but not to the K(d)/Pol(140-148) epitope. Different antigens transcribed from alternative reading frames of a single sequence in a DNA vaccine can thus efficiently prime multispecific T cell responses.