BackgroundHepatitis B virus infection remains an important global health concern despite the availability of safe and effective prophylactic vaccines. Limitations to these vaccines include requirement for refrigeration and three immunizations thereby restricting use in the developing world. A new nasal hepatitis B vaccine composed of recombinant hepatitis B surface antigen (HBsAg) in a novel nanoemulsion (NE) adjuvant (HBsAg-NE) could be effective with fewer administrations.Methodology and Principal FindingsPhysical characterization indicated that HBsAg-NE consists of uniform lipid droplets (349+/−17 nm) associated with HBsAg through electrostatic and hydrophobic interactions. Immunogenicity of HBsAg-NE vaccine was evaluated in mice, rats and guinea pigs. Animals immunized intranasally developed robust and sustained systemic IgG, mucosal IgA and strong antigen-specific cellular immune responses. Serum IgG reached ≥106 titers and was comparable to intramuscular vaccination with alum-adjuvanted vaccine (HBsAg-Alu). Normalization showed that HBsAg-NE vaccination correlates with a protective immunity equivalent or greater than 1000 IU/ml. Th1 polarized immune response was indicated by IFN-γ and TNF-α cytokine production and elevated levels of IgG2 subclass of HBsAg-specific antibodies. The vaccine retains full immunogenicity for a year at 4°C, 6 months at 25°C and 6 weeks at 40°C. Comprehensive pre-clinical toxicology evaluation demonstrated that HBsAg-NE vaccine is safe and well tolerated in multiple animal models.ConclusionsOur results suggest that needle-free nasal immunization with HBsAg-NE could be a safe and effective hepatitis B vaccine, or provide an alternative booster administration for the parenteral hepatitis B vaccines. This vaccine induces a Th1 associated cellular immunity and also may provide therapeutic benefit to patients with chronic hepatitis B infection who lack cellular immune responses to adequately control viral replication. Long-term stability of this vaccine formulation at elevated temperatures suggests a direct advantage in the field, since potential excursions from cold chain maintenance could be tolerated without a loss in therapeutic efficacy.

The currently available commercial human anthrax vaccine requires multiple injections for efficacy and has side effects due to its alum adjuvant. These factors limit its utility when immunizing exposed populations in emergent situations. We evaluated a novel mucosal adjuvant that consists of a nontoxic, water-in-oil nanoemulsion (NE). This material does not contain a proinflammatory component but penetrates mucosal surfaces to load antigens into dendritic cells. Mice and guinea pigs were intranasally immunized with recombinant Bacillus anthracis protective antigen (rPA) mixed in NE as an adjuvant. rPA-NE immunization was effective in inducing both serum anti-PA immunoglobulin G (IgG) and bronchial anti-PA IgA and IgG antibodies after either one or two mucosal administrations. Serum anti-PA IgG2a and IgG2b antibodies and PA-specific cytokine induction after immunization indicate a Th1-polarized immune response. rPA-NE immunization also produced high titers of lethal-toxin-neutralizing serum antibodies in both mice and guinea pigs. Guinea pigs nasally immunized with rPA-NE vaccine were protected against an intradermal challenge with ϳ1,000 times the 50% lethal dose (ϳ1,000؋ LD 50 ) of B. anthracis Ames strain spores (1.38 ؋ 10 3 spores), which killed control animals within 96 h. Nasal immunization also resulted in 70% and 40% survival rates against intranasal challenge with 10؋ LD 50 and 100؋ LD 50 (1.2 ؋ 10 6 and 1.2 ؋ 10 7 ) Ames strain spores. Our results indicate that NE can effectively adjuvant rPA for intranasal immunization. This potentially could lead to a needle-free anthrax vaccine requiring fewer doses and having fewer side effects than the currently available human vaccine.

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 dendritic cells. Here, we demonstrate that protein antigens formulated in unique oil-in-water nanoemulsions results in unique trans-cellular antigen uptake in ciliated nasal epithelial cells, leading to delivery into nasal associated lymphoid tissue. Nanoemulsion 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-β, 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.

Development of effective intranasal vaccines is of great interest due to their potential to induce both mucosal and systemic immunity. Here we produced oil-in-water nanoemulsion (NE) formulations containing various cationic and nonionic surfactants for use as adjuvants for the intranasal delivery of vaccine antigens. NE induced immunogenicity and antigen delivery are believed to be facilitated through initial contact interactions between the NE droplet and mucosal surfaces which promote prolonged residence of the vaccine at the site of application, and thus cellular uptake. However, the details of this mechanism have yet to be fully characterized experimentally. We have studied the physicochemical properties of the NE droplet surfactant components and demonstrate that properties such as charge and polar head group geometry influence the association of the adjuvant with the mucus protein, mucin. Association of NE droplets with mucin in vitro was characterized by various biophysical and imaging methods including dynamic light scattering (DLS), zeta potential (ZP), and surface plasmon resonance (SPR) measurements as well as transmission electron microscopy (TEM). Emulsion surfactant compositions were varied in a systematic manner to evaluate the effects of hydrophobicity and polar group charge/size on the NE-mucin interaction. Several cationic NE formulations were found to facilitate cellular uptake of the model antigen, ovalbumin (OVA), in a nasal epithelial cell line. Furthermore, fluorescent images of tissue sections from mice intranasally immunized with the same NEs containing green fluorescent protein (GFP) antigen demonstrated that these NEs also enhanced mucosal layer penetration and cellular uptake of antigen in vivo. NE-mucin interactions observed through biophysical measurements corresponded with the ability of the NE to enhance cellular uptake. Formulations that enhanced antigen uptake in vitro and in vivo also led to the induction of a more consistent antigen specific immune response in mice immunized with NEs containing OVA, linking NE-facilitated mucosal layer penetration and cellular uptake to enhancement of the immune response. These findings suggest that biophysical measurement of the mucoadhesive properties of emulsion based vaccines constitutes an effective in vitro strategy for selecting NE candidates for further evaluation in vivo as mucosal adjuvants.