The Epstein Barr virus (EBV) is one of the predominant tumor viruses in humans, but so far no therapeutic or prophylactic vaccination against this transforming pathogen is available. We demonstrated that heterologous prime-boost vaccination with the nuclear antigen 1 of EBV (EBNA1) either targeted to the DEC205 receptor on dendritic cells or expressed from a recombinant modified vaccinia virus Ankara (MVA) vector improved priming of antigen-specific CD4⁺ T-cell help. This help supported the expansion and maintenance of EBNA1 specific CD8⁺ T cells that are most efficiently primed by recombinant adenoviruses that encode EBNA1. These combined CD4⁺ and CD8⁺ T-cell responses protected from EBNA1 expressing T and B cell lymphomas, including lymphoproliferations that emerge spontaneously after EBNA1 expression. In particular the heterologous EBNA1-expressing adenovirus, boosted by EBNA1-encoding MVA vaccination, demonstrated protection as prophylactic and therapeutic treatment of the respective lymphoma challenges. Therefore, we suggest that such heterologous prime-boost vaccinations should be further explored for clinical development against EBV-associated malignancies as well as symptomatic primary EBV infection.

Background: The clinical benefit of immune checkpoint blockade (ICB) therapy is often limited by the lack of pre-existing CD8⁺ T cells infiltrating the tumor. In principle, CD8⁺ T-cell infiltration could be promoted by therapeutic vaccination. However, this remains challenging given the paucity of vaccine platforms able to induce the strong cytotoxic CD8⁺ T-cell response required to reject tumors. A therapeutic cancer vaccine that induces a robust cytotoxic CD8⁺ T-cell response against shared tumor antigens and can be combined with ICB could improve the outcome of cancer immunotherapy. Methods: Here, we developed a heterologous prime-boost vaccine based on a chimpanzee adenovirus (ChAdOx1) and a modified vaccinia Ankara (MVA) encoding MAGE-type antigens, which are tumor-specific shared antigens expressed in different tumor types. The mouse MAGE-type antigen P1A was used as a surrogate to study the efficacy of the vaccine in combination with ICB in murine tumor models expressing the P1A antigen. To characterize the vaccine-induced immune response, we performed flow cytometry and transcriptomic analyses. Results: The ChAdOx1/MVA vaccine displayed strong immunogenicity with potent induction of CD8⁺ T cells. When combined with anti-Programmed Cell Death Protein 1 (PD-1), the vaccine induced superior tumor clearance and survival in murine tumor models expressing P1A compared with anti-PD-1 alone. Remarkably, ChAdOx1/MVA P1A vaccination promoted CD8⁺ T-cell infiltration in the tumors, and drove inflammation in the tumor microenvironment, turning ‘cold’ tumors into ‘hot’ tumors. Single-cell transcriptomic analysis of the P1A-specific CD8⁺ T cells revealed an expanded population of stem-like T cells in the spleen after the combination treatment as compared with vaccine alone, and a reduced PD-1 expression in the tumor CD8⁺ T cells. Conclusions: These findings highlight the synergistic potency of ChAdOx1/MVA MAGE vaccines combined with anti-PD-1 for cancer therapy, and establish the foundation for clinical translation of this approach. A clinical trial of ChadOx1/MVA MAGE-A3/NY-ESO-1 combined with anti-PD-1 will commence shortly.

Background: Melanoma antigen gene (MAGE)-type antigens are promising targets for cancer immunotherapy as they are expressed in cancer cells but not in normal tissues, except for male germline cells. The mouse P1A antigen shares this MAGE-type expression pattern and has been used as a target antigen in preclinical tumor models aiming to induce antitumor CD8⁺ T-cell responses. However, so far only one MHC I-restricted P1A epitope has been identified. It is presented by H-2L^d in mice of the H-2^d genetic background such as DBA/2 and BALB/c. Given the availability of multiple genetically altered strains of mice in the C57BL/6 background, it would be useful to define P1A T-cell epitopes presented by the H-2^b haplotype, to facilitate more refined mechanistic studies. Methods: We employed a heterologous prime-boost vaccination strategy based on a chimpanzee adenovirus (ChAdOx1) and a modified vaccinia Ankara (MVA) encoding P1A, to induce P1A-specific T-cell responses in C57BL/6 mice. Vaccine-induced responses were measured by intracellular cytokine staining and multiparameter flow cytometry. We mapped the immunogenic CD8 epitope and cloned the cognate T-cell receptor (TCR), which we used for adoptive cell therapy. Results: ChAdOx1/MVA-P1A vaccination induces a strong P1A-specific CD8⁺ T-cell response in C57BL/6 mice. This response is directed against a single 9-amino acid peptide with sequence FAVVTTSFL, corresponding to P1A amino acids 43–51. It is presented by H-2D^b. P1A vaccination, especially with ChAdOx1 administered intramuscularly and MVA delivered intravenously, protected mice against P1A-expressing EL4 (EL4.P1A) tumor cell challenge. We identified and cloned four TCRs that are specific for the H-2D^b-restricted P1A_43-51 peptide. T cells transduced with these TCRs recognized EL4.P1A but not MC38.P1A and B16F10.P1A tumor cells, likely due to differences in the proteasome subtypes present in these cells. Adoptive transfer of these T cells in mice bearing EL4.P1A tumors reduced tumor growth and increased survival. Conclusions: We identified the first CD8⁺ T-cell epitope of the MAGE-type P1A tumor antigen presented in the H-2^b background. This opens new perspectives for mechanistic studies dissecting MAGE-type specific antitumor immunity, making use of the wealth of genetically altered mouse strains available in the C57BL/6 background. This should facilitate the advancement of specific cancer immunotherapies.

Therapeutic cancer vaccines, whether based on neoantigens or shared antigens, will likely be given in the clinic together with the standard of care, which often comprises immune checkpoint blockade therapy and chemotherapy. It remains unclear, however, whether vaccines effectively synergize with chemotherapy. Here, we tested the combination of a heterologous prime-boost viral vector vaccine with chemotherapy (CarboTaxol) and anti-PD- 1. We show that this triple combination improves tumor control and survival in different murine tumor models. CarboTaxol, and also cyclophosphamide, acted as an immune adjuvant for the vaccines, enhancing tumor-specific CD8+ T-cell responses, irrespective of the presence of a tumor. These chemotherapies expanded stem-like T cell factor 1 (TCF1)+CD8+ T cells. Inhibition of the transcriptional activity of TCF1/β-catenin with a small molecule inhibitor abolished the immune adjuvant effect of CarboTaxol. This study sheds light on the new immunomodulatory roles of chemotherapies and holds promises for clinical testing of this combination strategy. Highlights The combination of CarboTaxol with viral vector cancer vaccines and anti-PD-1 promotes better tumor control, tumor clearance, and survival CarboTaxol increases TCF1 expression in CD8+ T cells and expands stem-like TCF1+CD8+ T cells CarboTaxol acts as an adjuvant for cancer vaccines irrespective of the presence of a tumor and this effect is mediated by TCF1/β-catenin activity