Post-PEGylated and crosslinked polymeric ssRNA nanocomplexes as adjuvants targeting lymph nodes with increased cytolytic T cell inducing properties

Potent adjuvants are highly demanded for most protein and peptides based vaccine candidates in clinical development. Recognition of viral single stranded (ss)RNA by innate toll-like receptors 7/8 in dendritic cells results in a cytokine environment supportive to the establishment of long lasting antibody responses and Th1 oriented T cell immunity. To fully exploit the immunestimulatory properties of ssRNA, it needs to be adequately formulated to ensure its optimal delivery to dendritic cells in the vaccine draining lymph nodes. In the present paper, we report on the design of ssRNA nanocomplexes formed by complexation of the cationic poly(carbonic acid 2-dimethylamino-ethyl ester 1-methyl-2-(2-methacryloylamino)-ethyl ester) (pHPMA-DMAE) based polymeric carrier and ssRNA. The resulting ssRNA nanocomplexes were subsequently PEGylated through copper-free click chemistry using PEG-bicyclo[6.1.0]nonyne (PEG-BCN) and cross-linked via disulfide bonds to increase their stability. The obtained near-neutral charged PEGylated ssRNA nanocomplexes (~150 nm) combined ssRNA protection with highly efficient delivery of ssRNA to DCs in the vaccine draining lymph nodes after subcutanuously administration. When co-administrated with a model antigen (soluble ovalbumin (OVA)), ssRNA nanocomplexes were far more efficient at inducing CD8 cytolytic T cells when compared to OVA co-adminstarted with naked ssRNA. Furthermore, IgG2c antibody titers, indicative of Th1 skewed T cell responses, were >10 times increased by complexing ssRNA into the PEGylated nanocomplexes. This study highlights the potential of post-functionalizing ssRNA nanocomplexes by copper-free click chemistry and these findings indcate that this potent ssRNA adjuvant may profoundly improve the efficacy of a variety of vaccines requiring Th1-type immunity.