Measles Vaccines Designed for Enhanced CD8+ T Cell Activation

Abstract

Priming and activation of CD8⁺ T cell responses is crucial to achieve anti-viral and anti-tumor immunity. Live attenuated measles vaccine strains have been used successfully for immunization for decades and are currently investigated in trials of oncolytic virotherapy. The available reverse genetics systems allow for insertion of additional genes, including heterologous antigens. Here, we designed recombinant measles vaccine vectors for priming and activation of antigen-specific CD8⁺ T cells. For proof-of-concept, we used cytotoxic T lymphocyte (CTL) lines specific for the melanoma-associated differentiation antigen tyrosinase-related protein-2 (TRP-2), or the model antigen chicken ovalbumin (OVA), respectively. We generated recombinant measles vaccine vectors with TRP-2 and OVA epitope cassette variants for expression of the full-length antigen or the respective immunodominant CD8⁺ epitope, with additional variants mediating secretion or proteasomal degradation of the epitope. We show that these recombinant measles virus vectors mediate varying levels of MHC class I (MHC-I)-restricted epitope presentation, leading to activation of cognate CTLs, as indicated by secretion of interferon-gamma (IFNγ) in vitro. Importantly, the recombinant OVA vaccines also mediate priming of naïve OT-I CD8⁺ T cells by dendritic cells. While all vaccine variants can prime and activate cognate T cells, IFNγ release was enhanced using a secreted epitope variant and a variant with epitope strings targeted to the proteasome. The principles presented in this study will facilitate the design of recombinant vaccines to elicit CD8⁺ responses against pathogens and tumor antigens.

Keywords: CD8+ T cell activation; T cell priming; cancer immunotherapy; measles; oncolytic virus; vaccination.

Figure 1

Measles vaccine viruses encoding TRP-2 or ovalbumin (OVA) activate antigen-specific T cells. (a) Genome schematics of measles Schwarz vaccine strain viruses encoding TRP-2 (MeVac TRP-2, left panel) and ovalbumin (MeVac OVA, right panel). (b) IFNγ ELISpot assay. MC38-hCD46 cells were infected at MOI 3 with MeVac encoding the TRP-2 or OVA antigen or with unmodified MeVac and used as target cells for cytotoxic T lymphocytes (CTLs) which recognize the respective antigen-derived CTL epitope. Synthetic peptides and B16-OVA-hCD46 cells which express both antigens were used as controls. Representative results from one of three independent experiments are shown (upper panels: spot counts, lower panels: magnification of 96-well ELISpot plate). N, P, M, F, H, and L: Measles virus nucleocapsid, phosphoprotein, matrix, fusion, hemagglutinin, and large (polymerase) genes.

Figure 2

Measles vaccine viruses with epitope cassette variants mediate MHC-I restricted epitope presentation. (a) MC38-hCD46 cells were infected with MeVac OVA or unmodified MeVac at MOI 3. Twenty-four hours after infection, flow cytometry was performed with an antibody specific for OVA aa257–264 (SIINFEKL) presented by H-2K^b. (b) Genome schematic of measles Schwarz vaccine strain viruses with epitope cassette (EC) variants. (c) Epitope cassette variants for the TPR-2 (top panel) and ovalbumin (bottom panel) CTL epitopes. (d) MC38-hCD46 cells were infected with MeVac encoding epitope cassette variants or unmodified MeVac at MOI 3 in triplicates. Twenty-four hours after infection, flow cytometry was performed with an antibody specific for SIINFEKL presented by H-2K^b. Mean values of triplicates and 95% confidence intervals from one representative of three independent experiments are shown.

Figure 3

Measles vaccine viruses with epitope cassette variants activate cognate CD8⁺ T cells. MC38-hCD46 cells were infected with indicated MeVac variants at MOI 3 and used as target cells for CTLs which recognize the respective epitope, (a): SVYDFFVWL, (b) and (c): SIINFEKL. After 18 h of co-culture, IFNγ ELISpot assays were performed. Representative results from one of three independent experiments are shown.

Figure 4

Dendritic cells exposed to measles vaccine viruses with epitope cassette variants mediate epitope presentation by H-2K^b and activate cognate T cells. DC2.4 cells were inoculated with recombinant viruses encoding epitope cassette variants at MOI 3. Where indicated, viruses were inactivated by UV irradiation prior to inoculation. (a) After 24 h, H-2K^b-SIINFEKL was detected by flow cytometry with a PE-labeled antibody. Results from three independent experiments are shown with bars indicating mean values for each condition. (b) After 24 h, co-cultures with SIINFEKL-specific CTLs were established and IFNγ ELISpot assay was performed. Bars indicate mean values of six replicates for each condition. One representative of three independent experiments with non-inactivated viruses is shown.

Figure 5

Dendritic cells exposed to measles vaccine viruses with epitope cassette variants can prime cognate T cells. DC2.4 cells were inoculated with measles encoding epitope cassette variants. After 24 h, co-cultures with naïve T cells from OT-I mice were established and IFNγ ELISpot assay was performed. Bars indicate mean value of six replicates for each condition. One representative of three independent experiments is shown.