Replication-incompetent viral vaccine vectors ChAdOx1 and MVA as tools for evaluating T-cell responses to naturally processed antigens in vitro

• Safer viral vectors ChAdOx1 and MVA assess T-cell responses in vitro effectively • ChAdOx1 and MVA avoid high biosafety requirements of infectious viral agents • These viral vectors enable immune studies in resource-limited, low-biosafety settings Assessing T-cell responses is critical for vaccine development. In vitro methods using SARS-CoV-2 or recombinant vaccinia virus with B cells effectively activate T-cells but require stringent biosafety conditions. As an alternative, we explored attenuated, replication-incompetent viral vectors, such as modified vaccinia Ankara (MVA) and chimpanzee adenoviral vectors (ChAdOx1 and ChAdOx2). These vectors successfully transduced B cells, as confirmed by GFP expression. B cells transduced with ChAdOx1 nCoV-19 (encoding SARS-CoV-2 Spike) activated autologous CD8⁺ and CD4⁺ T-cells. Similarly, B cells transduced with MVA encoding Spike activated autologous CD4⁺ T-cells. Our findings provide proof-of-concept support for the use of these safer viral vectors in in vitro studies of vaccine-induced cellular immunity. Proposed method diagram. For a pathogen of interest, such as a pandemic or pandemic-prone pathogen, blood samples are collected from either a) convalescent patients who have recently recovered from an infection with the pathogen (3 to 6 weeks, allowing for recovery), or b) vaccinated individuals who have received either licensed vaccines or are participating in clinical trials evaluating the efficacy of novel interventions (4 to 8 weeks). From such samples, c) PBMCs are isolated and separated into B cells and T-cells (within a day after collection). Subsequently, d) the B cells are immortalised using EBV (2 to 3 weeks), and e) the T-cells are incubated with peptide pools derived from antigens of the pathogen under investigation (same or following day), and f) sorted as antigen-specific CD4+ and CD8+ T-cells (within 2 to 4 days). For the study of natural antigen processing, g) EBV-immortalised B cells are transduced with either ChAdOx1 or MVA viral vectors engineered to carry the genetic sequence of the antigen of interest (16 to 48 h). Following an overnight incubation, h) transduced B cells are co-cultured with either the antigen-specific CD4+ or CD8+ T-cells, and i) the phenotype of activation assessed by flow cytometry, either as j) CD4+IFN-γ+TNF-α+, or k) CD8+IFN-γ+TNF-α+ T-cells, the later steps of the protocol being performed between 4 and 24 h. Figure created with Biorender.