There is a need for more effective therapy for chronic virus infections. A principle natural mechanism for elimination of virus-infected host cells is activation of viral antigen-specific cytotoxic T lymphocytes (CTL). In an effort to develop methods of inducing virus-specific CTL responses that might be utilized in therapy of virus infections, we have investigated the effect of B7, a costimulatory factor for T-cell activation. In this study we show that delivery of genes encoding human B7-1 and a viral antigen in the same recombinant viral vector to cells of mice induces a greater viral antigen-specific CTL response than does similar delivery of the viral antigen gene alone. Two recombinant adenovirus vectors were constructed with the foreign genes inserted in the early region 3. One of them (Ad1312) directed expression of the surface antigen gene of hepatitis B virus (HBS); the other (Adl310) directed coexpression of HBS and human B7-1 (CD80) by means of an internal ribosomal entry site placed between the two coding sequences. When inoculated into BALB/c mice, both vectors induced a viral surface antigenspecific CTL response. The response induced by Adl310 was stronger than that by Ad1312 as measured by a chromium release assay for CTL activity and limiting dilution analysis for CTL precursor frequency, indicating that the B7-1 gene co-delivered with the HBS gene had an enhancing effect on the CTL response against surface antigen. Adl310 also induced a higher titer of antibody against surface antigen than did Ad1312. This result suggests that expression of a costimulatory protein and a viral antigen in the same cells in vivo induces stronger immune responses than expression of the antigen alone. This could be a novel strategy for development of both preventive and therapeutic vaccines against infectious agents.Activation of naive T cells requires not only recognition of an antigen-major histocompatibility complex on the surface of antigen-presenting cells (APC) by the T-cell receptor but also costimulatory signals, which can be provided by spleen cells of immunized mice (1). One such signal has been shown to result from interaction of costimulatory factors on the surface of APCs with specific molecules on the surface of T cells. Costimulatory factors on professional APCs include the B7-1 protein (CD80), which specifically interacts with the CD28 and CTLA-4 proteins on the surface of T cells resulting in a costimulatory signal for T-cell activation (2-9). B7-1 appears to be expressed only by professional APC (macrophages, dendritic cells, and activated B cells) and not other cells in vivo (3,10,11).The experiments described here were done to determine whether the cytotoxic T lymphocyte (CTL) response and antibody response are enhanced when cells expressing a foreign antigen in vivo are made to also express B7-1. This was done by delivering both genes for a viral antigen (hepatitis B surface antigen or HBsAg) and B7-1 in a recombinant viral vector to cells of BALB/c mice and comparing the immune response...