Immunogenicity of two recombinant DNA COVID-19 vaccines in young and aged mice.

Background: SARS-CoV-2 emerged in December 2019 and spread rapidly around the world. Vaccination is the most effective way to control the pandemic morbidity and mortality. While most of the currently available vaccines against COVID-19 have shown high efficacy against the original strain of SARS-CoV-2, their effectiveness has declined due to the emergence of new variants and diminished immunity remains a major threat, especially in older individuals. Therefore, the development of safe and effective vaccines that can be rapidly adapted to new SARS-CoV-2 variants represents an urgent health priority. We assessed the immunogenicity of two DNA vaccines against SARS-CoV-2 variants: pVAX-S1-TM-D614G and pVAX-S1-TM-INDUK. Methods: pVAX-S1-TM-D614G, encoding the S1 spike subunit in fusion with the transmembrane region, that allows protein trimerization as predicted by in silico analysis, was constructed by recombinant DNA technologies; the dominant D614G substitution was introduced by a PCR-based mutagenesis protocol. pVAX-S1-TM- INDUK was obtained by the insertion of additional key mutations from Delta (E484Q and L452R) and Alpha (N501Y and A570D) variants. Antigen expression was verified in vitro by immunofluorescence assay. To test the immunogenicity of pVAX-S1-TM- D614G and pVAX-S1-TM-INDUK, they were administered, via intramuscular injection followed by electroporation, in young and aged mice. The elicited immune responses were monitored for 6 months. Results: pVAX-S1-TM-D614G and pVAX-S1-TM-INDUK were first validated in vitro: a robust expression and membrane localization of antigenic proteins was demonstrated on transiently transfected HEK-293 cells. Our candidates DNA vaccines were then tested in vivo in both young (11 weeks of age) and aged (20 months of age) C57BL/6 mice. When delivered by electroporation, they were able to trigger a significant anti-SARS-CoV-2 antibody production in immunized mice, although antibody titer declined 6 months after the second dose, especially in aged animals. Of note, a third booster dose, given at 6 months from the last vaccination, significantly increased the magnitude of humoral immunity, suggesting that immune recall can improve antibody durability. Moreover, we optimized a lipid nanoparticle formulation, we called LNP15, to encapsulate DNA plasmids by microfluidic technology. Preliminary in vitro and in vivo results obtained with a prototype DNA vaccine, indicate that LNP15 can successfully encapsulate DNA vaccines for their easier administration. Conclusions: We developed two recombinant DNA vaccines (pVAX-S1-TM-D614G and pVAX-S1-TM-INDUK) against SARS-CoV-2 variants, able to elicit a significant anti-Spike antibody response in both young and aged mice. Although the humoral response declined within 6 months, a booster dose can efficiently recall immune memory and reverse anti-SARS-CoV-2 antibody waning even in aged population. Moreover, LNP15 formulation might permit to successfully deliver candidate DNA vaccines by intramuscular injection without electroporation. Given that DNA vaccines can be easily adapted in response to new variants, are cheaper and more stable than currently approved vaccines, they represent a promising strategy to achieve global immunization.