Humoral and cellular immune response to RNA immunization with flavivirus replicons derived from tick-borne encephalitis virus

Abstract

A new vaccination principle against flaviviruses, based on a tick-borne encephalitis virus (TBEV) self-replicating noninfectious RNA vaccine that produces subviral particles, has recently been introduced (R. M. Kofler, J. H. Aberle, S. W. Aberle, S. L. Allison, F. X. Heinz, and C. W. Mandl, Proc. Natl. Acad. Sci. USA 7:1951-1956, 2004). In this study, we evaluated the potential of the self-replicating RNA vaccine in mice in comparison to those of live, attenuated vaccines and a formalin-inactivated whole-virus vaccine (ImmunInject). For this purpose, mice were immunized using gene gun-mediated application of the RNA vaccine and tested for CD8+ T-cell responses, long-term duration, neutralizing capacity, and isotype profile of specific antibodies and protection against lethal virus challenge. We demonstrate that the self-replicating RNA vaccine induced a broad-based, humoral and cellular (Th1 and CD8+ T-cell response) immune response comparable to that induced by live vaccines and that it protected mice from challenge. Even a single immunization with 1 microg of the replicon induced a long-lasting antibody response, characterized by high neutralizing antibody titers, which were sustained for at least 1 year. Nevertheless, it was possible to boost this response further by a second injection with the RNA vaccine, even in the presence of a concomitant CD8+ T-cell response. In this way it was possible to induce a balanced humoral and cellular immune response, similar to infection-induced immunity but without the safety hazards of infectious agents. The results also demonstrate the value of TBEV replicon RNA for inducing protective long-lasting antiviral responses.

FIG. 1.

IFN-γ expression-based detection of CD8⁺ T-cell responses. Groups of four BALB/c mice were inoculated with the replicon mutant C(Δ28-89)-S or live, attenuated viruses [3′Δ10847 and C(Δ28-43)] or with a formalin-inactivated TBEV vaccine (ImmunInject). One group of mice immunized with the replicon C(Δ28-89)-S or with ImmunInject was given a second injection 4 weeks after initial immunization. CD8⁺ T cells specifically recognizing TBEV-infected cells were visualized by IFN-γ staining. Intracellular IFN-γ staining was done after culturing the spleen cells from immunized mice with MHC class I⁺ BALB/c-p815 cells or mock-infected control cells. (A) Percentages of CD8⁺ T cells specifically recognizing TBEV-infected cells in individual mice. The squares represent the mean of duplicate samples obtained from individual mice; lines represent overall mean of all samples. (B) Representative set of flow cytometry data. Dot plots were generated by gating on CD3⁺ T lymphocytes. Percentages of IFN-γ⁺ cells within CD3⁺ CD8⁺ cells are indicated in the upper right quadrant.

FIG. 2.

Geometric mean (lines) and individual values (black dots) of TBEV-specific IgG antibody titers 8 weeks after initial immunization with replicon RNA C(Δ28-89)-S, live, attenuated viruses [3′Δ10847 and C(Δ28-43)], or the formalin-inactivated TBEV vaccine (ImmunInject), as determined by ELISA; one group of mice immunized with the replicon C(Δ28-89)-S or with ImmunInject was given a second injection 4 weeks after the initial immunization.

FIG. 3.

Long-term IgG antibody response at weeks 12, 24, and 52 after initial immunization with the mutant C(Δ28-89)-S, live, attenuated viruses [3′Δ10847 and C(Δ28-43)], or the formalin-inactivated TBEV vaccine (ImmunInject), as determined by ELISA; bars represent the mean of duplicate assays using pooled sera (n = 4). One group of mice immunized with the replicon C(Δ28-89)-S or with ImmunInject was given a second injection 4 weeks after the initial immunization.