Immunogenicity of High-resolution in situ structures of hantavirus glycoprotein tetramers

Goals--The goal of the immunogenicity experiments was to determine whether repRNA/LION™ formulations encoding ANDV glycoprotein precursor (GPC) constructs elicit robust humoral and cellular immune responses in mice. Specifically, to compare dose effects (1 µg vs 10 µg) and evaluate the magnitude and durability of Gn–Gc–specific IgG responses and T cell responses. These studies aimed to assess whether the structural antigen design translated into immunogenicity consistent with a viable vaccine candidate, and to determine whether strategies that improved virus-like particle (VLP) formation also improved immunogenicity. Methods--Mice were immunized with 1 µg or 10 µg of repRNA/LION™-formulated ANDV-GPC constructs, and sera were collected longitudinally (Days 14, 35, and 49). Gn–Gc–specific IgG responses were quantified by ELISA, with IgG concentrations and endpoint titers (defined as the reciprocal serum dilution exceeding background by ≥2 standard deviations) reported. Cellular responses were assessed by IFN-γ ELISpot using splenocytes stimulated with pooled ANDV-GPC peptides. Neutralization against rVSVs encoding ANDV-GnGc was also assessed. Results--Both 1 µg and 10 µg doses induced increasing Gn–Gc–specific serum IgG over time, with higher titers observed at the 10 µg dose and after boosting (Fig. S11A–C). IgG concentrations and endpoint titers significantly increased between early and later timepoints, demonstrating a boost-responsive humoral response. All constructs were immunogenic, with repGPC-DM-eVLP eliciting the highest titers of binding antibodies. By rVSV-ANDV-GPC neutralization assays, however, neutralizing antibody titers were equivalent between wild-type and DM-eVLP GPC. IFN-γ ELISpot analysis showed dose-dependent cellular immunity, with higher spot-forming units per 10⁶ splenocytes in the 10 µg group, indicating that the vaccine constructs elicited both antibody and antigen-specific T cell responses.