Live-Attenuated and Novel Non-Replicating Listeria Vaccines Encoding CMV Antigen Produce Persistent Functional Antiviral Immunity.

Background: We previously described a live-attenuated (L/A) Listeria monocytogenes (Lm)-based vaccine encoding murine CMV (MCMV) epitopes (Lm-MCMV) that effectively drives expansion of antiviral CD8+ T-cells in wild-type mice and following bone marrow transplantation (BMT). We now show similar efficacy with increased safety using a non-replicating killed but metabolically active Lm vaccine (KBMA-Lm-MCMV). Furthermore, we demonstrate that resulting antiviral T-cells persist long-term (>200 days) and exert functional antiviral activity.

Methods: The L/A Lm-MCMV vaccine is derived from a genetically defined Lm ΔactA/ΔinlB vaccine strain (

Results: Vaccination of wild-type C57BL/6 mice with 10⁷ cfu L/A Lm-MCMV (< 0.03 LD₅₀) induced anti-HGIRNASFI CD8+ T-cells to a peak of 9.7% (+/- 1.6%) of total CD8+ T-cells 7 days later. In BMT mice, L/A Lm-MCMV vaccination was delayed to day 21 post-transplant to reduce mortality, and lead to peak CTL responses of 17.6% of total CD8 within 7 days. Interestingly, the novel non-replicating KBMA vaccine could be administered immediately after BMT without mortality, but nonetheless antiviral CTLs still expanded to 16% of total CD8 by 7 days after BMT. At > 200 days after vaccination of wild-type or BMT mice, HGIRNASFI-specific CD8+ T-cells still accounted for 0.5 - 5% of all CD8+ T-cells. Interestingly, mice convalescent after a similarly remote MCMV infection (10⁶ pfu) had significantly lower levels of antiviral T-cells (0.1-1%; p < 0.05). Following either Lm-MCMV vaccination or MCMV infection the majority of tetramer-positive cells were CD44^hi and CD62L^hi, consistent with the central memory subset of CD8+ T-cells. Using In vivo CTL assays, Lm-MCMV vaccination produced specific anti-HGIRNASFI lytic activity averaging 96.7% (+/− 4.0%) at > 200 days after vaccination, which was similar to that seen after MCMV infection. Vaccination also significantly reduced viral loads by 46% (p < 0.026) following experimental MCMV infection.

Conclusions: Vaccination with the L/A or KBMA Lm-MCMV vaccine rapidly produced high levels of anti-MCMV CD8+ T-cells that persisted long-term (>200 days), rapidly cleared MCMV-antigen pulsed target cells, and significantly reduced MCMV replication in vivo. Since severely immunocompromised BMT recipients could be safely vaccinated with KBMA-Lm-MCMV immediately after transplant without mortality, significant levels of virus-specific CTLs could be reconstituted within 7 days of transplantation. Given the resulting high levels of durable antigen-specific lytic activity, this approach could represent a broadly applicable alternative to adoptive immunotherapy to prevent viral disease after transplantation.