Nanoparticulate vaccines can potentiate immune responses by site-specific drainage to lymph nodes (LNs). This approach may benefit from a nanoparticle engineering method with fine control over size and codelivery of antigen and adjuvant. Here, we applied the flash nanocomplexation (FNC) method to prepare nanovaccines via polyelectrolyte complexation of chitosan and heparin to coencapsulate the VP1 protein antigen from enterovirus 71, which causes hand-foot-mouth disease (HFMD), with tumor necrosis factor α (TNF) or CpG as adjuvants. FNC allows for reduction of the nanovaccine size to range from 90 to 130 nm with relatively narrower size distribution and a high payload capacity. These nanovaccines reached both proximal and distal LNs via subcutaneous injection and subsequently exhibited prolonged retention in the LNs. The codelivery induced strong immune activation toward a Th1 response in addition to a potent Th2 response, and conferred effective protection against lethal virus challenge comparable to that of an approved inactivated viral vaccine in mouse models of both passive and active immunization setting. In addition, these nanovaccines also elicited strong IgA titers, which may offer unique advantages for mucosal protection. This study addresses the issues of size control, antigen bioactivity retention, and biomanufacturing to demonstrate the translational potential of a subunit nanovaccine design.

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It remains unclear how the precise length of onedimensional nanovehicles influences the characters of vaccination.Here, a unimolecular nanovehicle with tailored size and aspect ratio (AR) is applied to deliver CpG oligodeoxynucleotide, a Toll-like receptor (TLR) 9 agonist, as an adjuvant of recombinant hepatitis B virus surface antigen (rHBsAg), for treating chronic hepatitis B (CHB). Cationic nanovehicles with fixed width (ca. 45 nm) but varied length (46 nm−180 nm), AR from 1 to 4, are prepared through controlled polymerization and are loaded with CpG by electrostatic interaction. We reveal that the nanoadjuvant with AR = 2 shows the highest retention in proximal lymph nodes. Importantly, it is more easily internalized into antigen-presenting cells and accumulates in the late endosome, where TLR9 is located. Such a nanoadjuvant exhibits the strongest immune response with rHBsAg to clear the hepatitis B virus in the CHB mouse model, showing that the AR of nanovehicles governs the efficiency of vaccination.

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CpG, an agonist of toll-like receptor 9 (TLR9), has become a novel adjuvant that substantially potentiates cellular immunity. However, this agonist may increase systemic toxicity by diffusing into blood after administration and is difficult to be internalized by immune cells to reach TLR9 located in endosomes as a result of the characteristics of negative charge of CpG. Here, we applied a scalable and controllable flash nanocomplexation technology to prepare nanoparticulate CpG adjuvant (npCpG), CpG encapsulated in a physical cross-linking network of protamine and TPP. The nanoadjuvant could redirect CpG into draining lymph nodes to reduce systemic diffusion to improve safety. Further, a combination of npCpG and influenza H1N1 hemagglutinin antigen showed excellent humoral and cellular immunity, evoking high levels of antibodies and cytokines and inducing a great expansion of splenocytes in immunized mice. Also, the nanoadjuvant combined with ovalbumin antigen led to a potent cytotoxic T-cell response, substantially inhibited tumor growth, and improved the survival rate of mice in a melanoma model. This study showed the universal performances of npCpG in infectious disease prevention and tumor immunotherapy to demonstrate the translational potential.