An enantiomer-dependent and modification-free DNA matrix as a novel adjuvant platform for subunit vaccines

We introduce a novel dynamic DNA supramolecular matrix material assembled from five unmodified, short DNA strands, serving as a safe and effective adjuvant platform. Our findings demonstrate that this DNA matrix material elicits robust humoral response in an enantiomer-dependent but CpG-independent manner, effectively generating potent neutralizing antibodies and conferring robust protection against diverse pathogen infections, including both SARS-CoV-2 and Streptococcus pneumoniae. Remarkably, DNA matrix exhibits minimal adverse effects in comparison to the commonly used aluminum material. In stark contrast to its soluble DNA components, the dynamic colloidal feature of DNA matrix material prolongs the in vivo retention of both DNA and antigen by spontaneously disintegrating into nanoparticles in hundreds of nanometers range, facilitating lymphatic-targeted transportation and accumulation. This process leads to a robust pro-inflammatory response in both vaccinated local tissue and draining lymph node (dLN), which in turn promotes the recruitment and activation of diverse immune cells, ultimately leading to a rapid and robust antigen-specific antibody response. Surprisingly, mechanistic investigation uncovers that the enhancing function of this DNA matrix material is not dependent on DNA-sensor machineries including cGAS, ZBP1, AIM2 and STING, relying instead on the TLR9-MyD88 signaling axis. Cell-lineage specific MyD88 knockout experiments underscore the critical role of this axis in DCs, but not in B cells and keratinocytes. Unexpectedly, the antibody-enhancing function of this DNA matrix material is also strongly dependent on the right-handed chirality of the building block DNA strands, forming D-DNA matrix. In marked contrast, the left-handed L-DNA matrix, despite inheriting identical physiochemical properties as a material and the ability to disintegrate into nanoparticles as D-DNA matrix, completely fails to promote immune activations in vitro and in vivo. Thus, this injectable, self-assembling, CpG- and modification-free DNA matrix material functions as a novel, all-in-one subunit vaccine adjuvant platform, opening up promising avenues in next-generation vaccine design.