Formulation, high throughput in vitro screening and in vivo functional characterization of nanoemulsion-based intranasal vaccine adjuvants.

Vaccine adjuvants have been reported to induce both mucosal and systemic immunity when applied to mucosal surfaces and this dual response appears important for protection against certain pathogens. Despite the potential advantages, however, no mucosal adjuvants are currently approved for human use. Evaluating compounds as mucosal adjuvants is a slow and costly process due to the need for lengthy animal immunogenicity studies. We have constructed a library of 112 intranasal adjuvant candidate formulations consisting of oil-in-water nanoemulsions that contain various cationic and nonionic surfactants. To facilitate adjuvant development we first evaluated this library in a series of high-throughput, in vitro assays for activities associated with innate and adaptive immune activation in vivo. These in vitro assays screened for the ability of the adjuvant to bind to mucin, induce cytotoxicity, facilitate antigen uptake in epithelial and dendritic cells, and activate cellular pathways. We then sought to determine how these parameters related to adjuvant activity in vivo. While the in vitro assays alone were not enough to predict the in vivo adjuvant activity completely, several interesting relationships were found with immune responses in mice. Furthermore, by varying the physicochemical properties of the surfactant components (charge, surfactant polar head size and hydrophobicity) and the surfactant blend ratio of the formulations, the strength and type of the immune response generated (TH1, TH2, TH17) could be modulated. These findings suggest the possibility of using high-throughput screens to aid in the design of custom adjuvants with unique immunological profiles to match specific mucosal vaccine applications.

Nanoemulsion mucosal adjuvant uniquely activates cytokine production by nasal ciliated epithelium and induces dendritic cell trafficking.

While the nasal mucosa is a potentially useful site for human immunization, toxin-based nasal adjuvants are generally unsafe and less effective in humans. Safe mucosal adjuvants that activate protective immunity via mucosal administration are highly dependent on barrier antigen sampling by epithelial and DCs. Here, we demonstrate that protein antigens formulated in unique oil-in-water nanoemulsions (NEs) result in distinctive transcellular antigen uptake in ciliated nasal epithelial cells, leading to delivery into nasal associated lymphoid tissue. NE formulation also enhances MHC class II expression in epithelial cells and DC activation/trafficking to regional lymphoid tissues in mice. These materials appear to induce local epithelial cell apoptosis and heterogeneous cytokine production by mucosal epithelial cells and mixed nasal tissues, including G-CSF, GM-CSF, IL-1a, IL-1b, IL-5, IL-6, IL-12, IP-10, KC, MIP-1a, TGF-ß, and TSLP. This is the first observation of a nasal adjuvant that activates calreticulin-associated apoptosis of ciliated nasal epithelial cells to generate broad cytokine/chemokine responses in mucosal tissue.