FreeStyle 293-F cells were split to a density of 0.8 x 10^6 cells/mL at least one hour before transfection. Cells were transfected with 90 mg plasmid DNA by 200 mL of cell culture (2:1 ratio of heavy and light chain) using FectoPRO at a 1:1 DNA to FectoPRO ratio (Polyplus instructions). Transfected cells were incubated in a 37°C, 5% CO2 in shaking incubator for 5 to 7 days to allow for the expression and pairing of heavy and light chain gene products. Transfected cell culture supernatants were collected and filtered through 0.22 mM Steritop filters before loading onto protein A affinity columns using the AKTA start protein purification system. Following loading, samples were washed with 1X PBS then eluted with 100 mM glycine, pH 2.2 and immediately neutralized with 1 M Tris, pH 9.0. Elution fractions were concentrated using Amicon 30K Ultra-0.5 mL Centrifugal Filters and buffer-exchanged into PBS with PD-10 desalting columns. All purified proteins were validated for integrity and purity via SDS-PAGE and stored at -80°C until use.

Subunit vaccines typically require co-administration with an adjuvant to elicit protective immunity, adding development hurdles that can impede rapid pandemic responses. To circumvent the need for adjuvant in a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) subunit vaccine, we engineer a thermostable immunotargeting vaccine (ITV) that leverages the pan-HLA-DR monoclonal antibody 44H10 to deliver the viral spike protein receptor-binding domain (RBD) to antigen-presenting cells. X-ray crystallography shows that 44H10 binds to a conserved epitope on HLA-DR, providing the basis for its broad HLA-DR reactivity. Adjuvant-free ITV immunization in rabbits and ferrets induces robust anti-RBD antibody responses that neutralize SARS-CoV-2 variants of concern and protect recipients from SARS-CoV-2 challenge. We demonstrate that the modular nature of the ITV scaffold with respect to helper T cell epitopes and diverse RBD antigens facilitates broad sarbecovirus neutralization. Our findings support anti-HLA-DR immunotargeting as an effective means to induce strong antibody responses to subunit antigens without requiring an adjuvant.