Joyce A. Sutcliffe

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

ABSTRACT NB-1008 is a surfactant-stabilized soybean oil-in-water nanoemulsion (NE) adjuvant with influenza virus antigen incorporated into the NE by simple mixing. Intranasal administration of the antigen with NE adjuvant efficiently produces both mucosal and serum antibody responses as well as a robust cellular Th1 immune response. To demonstrate the adjuvant effect of the W805EC NE, a killed commercial influenza vaccine for intramuscular administration (Fluzone or Fluvirin) was mixed with the W805EC NE adjuvant and administered intranasally to naïve ferrets. After a single intranasal immunization, the adjuvanted influenza vaccine elicited elevated serum hemagglutination inhibition (HAI) geometric mean titers (GMTs) ranging from 196 to 905 for the three hemagglutinin (HA) antigens present in the vaccine, which are approximately 19- to 90-fold higher titers at 1/50 the standard intramuscular commercial nonadjuvanted influenza vaccine dose. Seroconversion rates of 67% to 100% were achieved against each of the three viral strains present. The adjuvanted nasal influenza vaccine also produced significant cross immunity to five other H3N2 influenza virus strains not present in the vaccine and produced sterile immunity after challenge with homologous live virus. No safety issues were observed in 249 ferrets receiving the adjuvanted influenza vaccine. These findings demonstrate the ability of W805EC NE to adjuvant nasally administered influenza vaccine and provide a basis for studying the intranasal W805EC-adjuvanted influenza vaccine in humans.

Efficacy, immunogenicity and stability of a novel intranasal nanoemulsion-adjuvanted influenza vaccine in a murine model.

Mutations of influenza virus increase concerns of worldwide epidemics resulting from the newly emergent strains. Current influenza vaccines are inefficient and require annual vaccinations. W805EC adjuvant is an oil-in-water emulsion composed of nanodroplets with an average diameter of approximately 400 nm. The nanoemulsion adjuvant has been used successfully to stimulate the immune response when mixed with several other antigens in animal models. In this study, W805EC nanoemulsion adjuvant activity was evaluated using nasal influenza vaccination in a murine model. Five to twenty percent W805EC adjuvant was used to inactivate influenza A/Puerto Rico/8/34/05 (H1N1). Mice immunized with the nanoemulsion adjuvanted influenza virus intranasally showed a robust specific humoral immune response as demonstrated using ELISA and HAI assays. Serum HAI titers were more than 104 following two vaccinations. Vaccinated mice were also protected against challenge with an LD80 of live influenza virus. Splenocytes from vaccinated mice were assayed for cytokine production following virus stimulation. The cytokine profile demonstrated a robust cellular immune response with enhanced Th1 and Th17 immunity that provided balanced immunity against both intracellular and extracellular forms of the virus. Additionally, the vaccine preparations showed minimal protein degradation but remained potent when stored at 4ºC for up to three months. This work demonstrates that W805EC nanoemulsion adjuvant can effectively enhance the immunogenicity of influenza hemagglutinin antigen. The nanoemulsion adjuvant can result in antigen sparing and cross-protection. The potential exists for a nasally administered influenza vaccine that may require little or no refrigerated storage.

NB-002, a Novel Nanoemulsion with Broad Antifungal Activity Against Dermatophytes, Other Filamentous Fungi and Candida albicans

ABSTRACT NB-002 is an oil-in-water emulsion designed for use for the treatment of skin, hair, and nail infections. The activity of NB-002 was compared to the activities of the available antifungal drugs against the major dermatophytes responsible for cutaneous infections, Trichophyton rubrum, Trichophyton mentagrophytes, Epidermophyton floccosum, and Microsporum spp., as well as 12 other genera of filamentous fungi. NB-002 consistently displayed fungicidal activity against all dermatophytes. The comparator compounds were either fungistatic or fungicidal, and for some strain-drug combinations, tolerance was observed. Assessment of the development of spontaneous resistance to NB-002 in different dermatophyte species yielded few stably resistant mutants. For filamentous nondermatophyte fungi, the MIC range varied from 0.06 to 0.5 μg/ml for Alternaria spp. to 2 to 8 μg/ml for Paecilomyes spp. NB-002 had activity against both azole-susceptible and -resistant Candida albicans yeast isolates, with MIC90s of 2 μg/ml, respectively, and minimum fungicidal concentrations at which 90% of isolates are inhibited of 4 and 8 μg/ml, respectively. The kinetics of the fungicidal activity of NB-002 against T. rubrum isolates were compared to those of the other antifungal drugs. NB-002 killed both mycelia and microconidia even when the fungal forms were dormant or not actively growing. Electron micrographs of mycelia and spores treated with NB-002 showed the significant disruption of the fungal structure. The in vitro broad coverage of NB-002 against filamentous fungi, dermatophytes, and C. albicans, as well as its rapid fungicidal activity, warrants further investigations to ascertain if NB-002 would be useful for the treatment of cutaneous mycoses.

In Vitro Antibacterial Activity of NB-003 against Propionibacterium acnes

ABSTRACT NB-003 and NB-003 gel formulations are oil-in-water nanoemulsions designed for use in bacterial infections. In vitro susceptibility of Propionibacterium acnes to NB-003 formulations and comparator drugs was evaluated. Both NB-003 formulations were bactericidal against all P. acnes isolates, including those that were erythromycin, clindamycin, and/or tetracycline resistant. In the absence of sebum, the MIC90s/minimum bactericidal concentrations (MBC90s) for NB-003, NB-003 gel, salicylic acid (SA), and benzoyl peroxide (BPO) were 0.5/2.0, 1.0/2.0, 1,000/2,000, and 50/200 μg/ml, respectively. In the presence of 50% sebum, the MIC90s/MBC90s of NB003 and BPOs increased to 128/1,024 and 400/1,600 μg/ml, respectively. The MIC90s/MBC90s of SA were not significantly impacted by the presence of sebum. A reduction in the MBC90s for NB-003 and BPO was observed when 2% SA or 0.5% BPO was integrated into the formulation, resulting in MIC90s/MBC90s of 128/256 μg/ml for NB003 and 214/428 μg/ml for BPO. The addition of EDTA enhanced the in vitro efficacy of 0.5% NB-003 in the presence or absence of 25% sebum. The addition of 5 mM EDTA to each well of the microtiter plate resulted in a >16- and >256-fold decrease in MIC90 and MBC90, yielding a more potent MIC90/MBC90 of ≤1/<1 μg/ml. The kinetics of bactericidal activity of NB-003 against P. acnes were compared to those of a commercially available product of BPO. Electron micrographs of P. acnes treated with NB-003 showed complete disruption of bacteria. Assessment of spontaneous resistance of P. acnes revealed no stably resistant mutant strains.