TLR9 activation is required for cytotoxic response elicited by baculovirus capsid display

MARÍA I. CRESPO; PAULA MOLINARI; GUIDO N. MOLINA; BELKYS MALETTO; OSCAR TABOGA; IGNACIO CEBRIÁN; GABRIEL MORON

Mar del Plata

Congreso; Reunión Anual de Sociedades de Biociencias 2022; 2022

SAIC-SAI-FAIC-SAFIS

The baculovirus (BV) infects lepidopteran as natural hosts and represents an efficient vector for vaccine development. We showed that BV expressing OVA in its capsid (BVOVA) improved the immune response by eliciting CD8+ T cell activation. BVs enter intact into subcellular compartments of dendritic cells (DC) where both viral surface proteins and capsid follow separate intracellular routes, allowing the viral capsid containing the foreign Ag to reach the cytosol. In this study, we analyzed the intracellular mechanisms involved in CD8+ T cell activation by BVOVA. We first evaluated if the BV GP64-fusogenic protein is required for OVA cross-presentation by pretreating BVOVA with an anti-GP64 neutralizing antibody. In this case, infected splenic DCs failed to activate the B3Z CD8+ T cell hibridoma, specific for the MHCI(Kb)-OVA257–264. Moreover, T cell activation was inhibited when DCs were treated with lactacystin, indicating that proteasome is required for efficiently cross-presentation. We then analyzed the dependence of TLR9 and STING signaling by infecting DCs with BVOVA in the presence of H151, a STING inhibitor, or IRS869, a TLR9 signaling inhibitor. Only the incubation with IRS869 blocked B3Z activation by DCs. We also evaluated whether TLR9 is required to induce proliferation of naïve CD8+ T cells using BVOVA-infected DCs from WT and TLR9-/- mice. We found that TLR9-/- DCs cannot activate naïve CD8+ T cells efficiently. Also, TLR9-/- mice fail to generate an OVA-specific cytotoxic response after BVOVA injection. Finally, using IFNAR-/- mice we observed type I IFN are not required for cytotoxic response induction. In this work, we found that the viral envelope fuses with the endosomalmembrane allowing the viral capsid exits to cytosol for proteasomal degradation. During this BVOVA intracellular trafficking, endosomal TLR9 signaling in DCs is triggered, thereby stimulating the presentation of the MHCI/OVA257-264 complex to CD8+ T cells, which activate into CTL.