Pre-Clinical Evaluation of a Novel Nanoemulsion-Based Hepatitis B Mucosal Vaccine

Abstract

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.

Intranasal Immunization of Ferrets with Commercial Trivalent Influenza Vaccines Formulated in a Nanoemulsion-Based Adjuvant

“…In other studies, when an NE adjuvant prototype was combined with purified antigens such as recombinant anthrax protective antigen (8) and HIV gp120, it elicited a Th1 systemic response with neutralizing serum antibodies and mucosal IgA when it was administered intranasally to mice or guinea pigs (9). W 80 5EC NE combined with hepatitis B virus surface antigen and administered intranasally produced an enhanced immune response and caused no inflammation in the nasal cavity, no histopathological changes in key organs, and no abnormal laboratory findings in safety studies performed with mice, rats, guinea pigs, and dogs (23). These data suggest a broad adjuvant activity for the W 80 5EC NE and an acceptable safety profile for this adjuvant.…”

“…W 80 5EC NE contains droplets of approximately 400 nm in diameter and has inherent antimicrobial activity (23). Studies with a prototype NE formulations showed that the NE can inactivate whole influenza virus and induce an immune response after nasal administration that protects mice from homologous influenza virus challenge (25).…”

“…Immunization with recombinant HIV gp120 and NE adjuvant produced a Th1 immune response and neutralizing antibodies in mice (9). Recently, a more optimized nanoemulsion formulation, W 80 5EC NE, was combined with hepatitis B virus surface antigen and produced a robust immune response when administered intranasally, without evidence of inflammation in the nasal cavity or key body organs, including the brain, in four animal species (23). These data suggest broad adjuvant activity and an acceptable safety profile for the W 80 5EC adjuvant.…”

Distinct Pathways of Humoral and Cellular Immunity Induced with the Mucosal Administration of a Nanoemulsion Adjuvant

“…We recently developed a mucosal nanoemulsion (NE) adjuvant, W 80 5EC, that does not contain known TLR or other innate receptor ligands but is able to enhance both humoral and cellular immunity (27,31,32). This NE adjuvant is simply an oil-in-water formulation of emulsified, highly refined soybean oil combined with nonionic and cationic surfactants and ethanol, and it is similar in size to many viruses that infect the nasal mucosa (32).…”

Nanotechnologies Applied in Biomedical Vaccines

“…The potent nanotoxoid formulations provide a viable anti-virulence approach to combat with microorganisms that contain membrane damaging toxins, such as Staphylococcus aureus and Group A streptococcal infections [34]; (2) Stable at Room Temperature The common adjuvant-Alum is known to cause irritation if it is used in the conventional hepatitis B vaccine. However, the use of needle-free nasal immunization with a combination of nanoemulsion and hepatitis B antigen was found to be tolerable, effective and safe without side effects [35]. The solvent extraction or evaporation from a W/O/W (water-in-oil-in water) emulsion is usually used to prepare the antigen-encapsulated nanoparticles [36].…”