Biodegradable Hyaluronic Acid Modified with Tetraglycine‑l‑octaarginine as a Safe Adjuvant for Mucosal Vaccination

We have been investigating the potential use of polymers modified with cell-penetrating peptides as an adjuvant for mucosal vaccination and have already developed nondegradable poly­(N-vinylacetamide-co-acrylic acid) (PNVA-co-AA) with which d-octaarginine, a typical cell-penetrating peptide, was grafted. Our previous murine infection experiments demonstrated that immunoglobulin G (IgG) and immunoglobulin A (IgA) were induced in systemic circulation and secreted on nasal mucosa, respectively, through 4-time nasal inoculations with a mixture of influenza viral antigens and d-octaarginine-linked PNVA-co-AA at 7-day intervals, and that immunized mice were perfectly protected from homologous virus infection. In the present study, we designed novel biodegradable polymers bearing cell-penetrating peptides from a perspective of clinical application. Hyaluronic acid whose glucuronic acid was modified with tetraglycine-l-octaarginine at a monosaccharide unit ratio of 30% was successfully developed. The hyaluronic acid derivative exhibited adjuvant activities identical to PNVA-co-AA bearing either d-octaarginine or tetraglycine-d-octaarginine under the above-mentioned inoculation schedule. We further found that there was no difference in humoral immunity between the 4-time inoculations at 7-day intervals and the 2-time inoculations at 28-day intervals. Intranasal IgA induced through the latter schedule with a smaller number of inoculations, which is clinically practical, exhibited cross-reactivity beyond the subtype of viral strains. In vitro toxicity studies demonstrated that the hyaluronic acid derivative was much less toxic than the corresponding PNVA-co-AA derivatives, and that both the polymers and their metabolites did not exhibit genotoxicity. Our results suggested that tetraglycine-l-octaarginine-linked hyaluronic acid would be a clinically valuable and safe adjuvant for mucosal vaccination.