Immunization with Live-Attenuated Listeria Encoding CMV Antigen Induces Extensive Expansion of CMV-Specific CD8+ T-Cells Following HSCT: An Alternative to Adoptive Antiviral Immunotherapy.

Background: Recipients of hematopoietic stem cell transplantation (HSCT) can be severely immunocompromised, predisposing to opportunistic infections including cytomegalovirus (CMV). While adoptive transfer of ex vivo expanded donor antiviral T-cells can prevent viral disease, this approach is not frequently used clinically because cell culture and selection procedures are lengthy, labor-intensive, and expensive. We describe an alternative approach. A single inoculation of a strongly immunogenic, but safe, live-attenuated, Listeria-based vaccine (Lm-MCMV) can rapidly drive extensive in vivo expansion of anti-murine CMV (MCMV) CD8+ T-cells during immune reconstitution following HSCT.

Methods: The Lm-MCMV vaccine is derived from a genetically defined Listeria monocytogenes ΔactAΔinlB vaccine strain (Brockstedt et al. PNAS in press) that is attenuated by 4-logs in a mouse virulence assay, as compared to wild-type Listeria. Lm ΔactAΔinlB was engineered to express and secrete the MCMV H-2^b immunodominant peptide HGIRNASFI within an ovalbumin scaffold. C57BL/6 (H-2^b; CD45.2) HSCT 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 simultaneously received 3x10⁷ splenocytes from PepBoy (H-2^b; CD45.1) donors that were either naive or immunized with 1x10⁷ colony forming units (cfu) Lm-MCMV on day -7. On day +21, selected groups were vaccinated with 1x10⁷ cfu Lm-MCMV, and sacrificed up to 14 days later. MCMV-specific CD8+ T-cells were quantified by intracellular cytokine and tetramer staining. Residual viable Lm-MCMV vaccine in the spleen, liver, and brain of BMT recipients was measured by plating on BHI agar media.

Results: No mortality occurred following vaccination. Viable Lm-MCMV, which peaked the day after immunization in the liver (avg = 6.1x10⁴ cfu) and spleen (avg = 1.4x10⁵ cfu) of HSCT recipients, were completely cleared within 5 days of inoculation. Viable Listeria were not isolated from brain. Without vaccination at day +21, none of the HSCT recipients had detectable anti-MCMV CD8+ T cells. In contrast, immunization with Lm-MCMV at day +21 lead to marked antiviral T-cell expansion in all groups. HGIRNASFI-specific CD8+ T-cells averaged 2.4% of total CD8+ T-cells in vaccinated TCD marrow recipients. Levels were significantly higher in recipients of TCD marrow plus naive (7.1%) or immune (19.0%) donor splenocytes.

Conclusions: Immunization of BMT mice with live-attenuated Lm-MCMV was safe. All HSCT recipients survived immunization and rapidly cleared viable Listeria. A single Lm-MCMV immunization during the period of immune reconstitution following HSCT was also effective, driving extensive antiviral T-cell expansion. In HSCT recipients of TCD marrow alone, vaccination produced levels of HGIRNASFI-specific CD8+ T-cells (2.4%) comparable to that seen with adoptive immunotherapy. Co-transplantation of naive or immune donor splenocytes lead to significantly higher levels of antiviral CD8+ T-cells following vaccination (7.1% and 19%, respectively). This approach could represent a broadly applicable alternative to adoptive immunotherapy. Future studies will focus on optimizing vaccination strategies, including use in allogeneic transplantation.