Live-Attenuated and Novel Non-Replicating Listeria Vaccines Encoding CMV Antigen Produce Persistent Functional Antiviral Immunity.
Authors: Roback John D. , Lezhava Levan J. , Luckett William , Dubensky Thomas W. , Hillyer Christopher D. , Giedlin Martin 
Published: April 2020

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 (&gt;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 (Brockstedt et al., PNAS101:13832, 2004) that is attenuated by 4-logs in a mouse virulence assay. The non-replicating KBMA vaccine is derived from ΔactA/ΔuvrAB bacterial mutants treated with amotosalen, a proprietary DNA-crosslinking psoralen. Both vaccines express the MCMV H-2b immunodominant peptide HGIRNASFI. C57BL/6 (H-2b) BMT recipients were conditioned with 11 Gy irradiation on day -1, and injected with 5x106 T cell depleted (TCD) C57BL/6 bone marrow cells on day 0. Selected mice received 3x107 splenocytes from syngeneic donors that were immunized 7 days previously with 107 colony forming units (cfu) Lm-MCMV. BMT mice were vaccinated with L/A Lm-MCMV on day 21 after transplant, or with KBMA-Lm-MCMV on days 1, 2, and 3 after transplant.

Results: Vaccination of wild-type C57BL/6 mice with 107 cfu L/A Lm-MCMV (&lt; 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 &gt; 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 &lt; 0.05). Following either Lm-MCMV vaccination or MCMV infection the majority of tetramer-positive cells were CD44hi and CD62Lhi, 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 &gt; 200 days after vaccination, which was similar to that seen after MCMV infection. Vaccination also significantly reduced viral loads by 46% (p &lt; 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 (&gt;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.