Quantitative and qualitative analyses of the immune responses induced by amultivalent minigene DNA vaccine

Vaccines containing minigenes - isolated antigenic epitopes encoded by shor t open reading frames - can, under certain circumstances, confer protective immunity upon the vaccinee. Here we evaluate the efficacy of the minigene vaccine approach using DNA immunization and find that, to be immunogenic, a minigene-encoded epitope requires a perfect "Kozak" translational initiati on region. Tn addition, using intracellular cytokine staining, we show that immunization with a plasmid encoding a full-length protein induces epitope -specific CD8(+) T cells which are detectable directly ex vivo, and constit ute similar to 2% of the vaccinee's splenic CD8(+) T cells. In contrast, su ch cells are undetectable directly ex vivo in recipients of a minigene vacc ine. Nevertheless, the minigene plasmid does induce a low number of epitope -specific CD8(+) T cells, which can be amplified to detectable levels by in vivo stimulation. Indeed, 4 days after in vivo stimulation (by virus infec tion), all vaccinated mice regardless of whether they had been vaccinated w ith the minigene or with the full-length gene - had similar numbers of epit ope-specific CD8(+) T cells. However, despite these strong responses at 4 d ays post-infection, recipients of the minigene vaccine showed no enhanced a bility to limit virus replication and dissemination. We therefore observe a dichotomy; minigene vaccinees are not protected, despite the presence of s trong virus-specific immune responses at 4 days post-challenge. We suggest that the protective benefits of vaccination exert themselves very soon - pe rhaps within minutes or hours - after virus challenge. If the vaccine-induc ed immune response is too low to achieve this early protective effect, viru s-specific T cells will expand rapidly, but ineffectually, leading to the s trong but non-protective response measured at 4 days post-infection. Thus. vaccine-induced immunity should be monitored very early in infection, since the extent to which these responses may Inter be amplified is largely irre levant to the protection observed. 2000 Elsevier Science Ltd. All rights re served.