Tarek Hamouda

A novel surfactant nanoemulsion with a unique non-irritant topical antimicrobial activity against bacteria, enveloped viruses and fungi.

A novel non-ionic surfactant nanoemulsion designated 8N8 has been tested for its biocidal activity. One percent 8N8 produced effective bactericidal activity against Bacillus cereus, Bacillus subtilis, Haemophilus influenzae, Neisseria gonorrhoeae, Streptococcus pneumoniae, and Vibrio cholerae in 15 minutes. In contrast, most enteric gram-negative bacteria were resistant to 8N8. One percent 8N8 was also virucidal within 15 minutes for all tested enveloped viruses, including Herpes simplex type 1, influenza A and vaccinia viruses. One percent 8N8 also demonstrated fungistatic activity on Candida albicans. The rapid and non-specific inactivation of vegetative bacteria and enveloped viruses, in addition to its fungistatic activity and low toxicity in experimental animals, makes 8N8 a potential candidate for use as a topical biocidal agent.

Antimicrobial mechanism of action of surfactant lipid preparations in enteric Gram-negative bacilli.

Two surfactant lipid preparations (SLPs) were investigated to determine their mechanism of antimicrobial action. 8N8, a water‐in‐oil emulsion, and W60C, a liposome, both have bactericidal activity against Gram‐positive bacteria and non‐enteric Gram‐negative bacteria. Additionally, W60C is bactericidal for enteric Gram‐negative bacilli when suspended in deionized water. Zeta potential measurements suggested that the resistance of Gram‐negative bacilli to 8N8 might be caused by ionic repulsion. Addition of 50 µmol l−1 ethylene diamine tetra acetic acid in 100 mmol l−1 Tris buffer to either SLPs yielded efficient bactericidal activity against Gram‐negative bacilli. This appeared to be due to disruption of the outer membrane and the chelation of divalent cations, as the addition of excess calcium inhibited the antimicrobial effect. Electron microscopy studies documented that 8N8 disrupts the bacterial cell wall, lysing the bacteria, while W60C fuses and internalizes within the cell, causing damage without immediate cell lysis. Understanding the mechanisms of action of these biocidal formulations will help to produce improved formulations with broader spectra of activity.

A novel surfactant nanoemulsion with broad-spectrum sporicidal activity against Bacillus species

Two nontoxic, antimicrobial nanoemulsions, BCTP and BCTP 401, have been developed. These emulsions are composed of detergents and oils in 80% water. BCTP diluted up to 1:1000 inactivated>90% of Bacillus anthracis spores in 4 h and was also sporicidal against three other Bacillus species. This sporicidal activity is due to disruption of the spore coat after initiation of germination without complete outgrowth. BCTP 401 diluted 1:1000 had greater activity than BCTP against Bacillus spores and had an onset of action of <30 min. Mixing BCTP or BCTP 401 with Bacillus cereus prior to subcutaneous injection in mice reduced the resulting skin lesion by 99%. Wound irrigation with BCTP 1 h after spore inoculation yielded a 98% reduction in skin lesion size, and mortality was reduced 3-fold. These nanoemulsion formulas are stable, easily dispersed, nonirritant, and nontoxic compared with other available sporicidal agents.

Prevention of Influenza Pneumonitis by Sialic Acid–Conjugated Dendritic Polymers

Influenza A viral infection begins by hemagglutinin glycoproteins on the viral envelope binding to cell membrane sialic acid (SA). Free SA monomers cannot block hemagglutinin adhesion in vivo because of toxicity. Polyvalent, generation 4 (G4) SA-conjugated polyamidoamine (PAMAM) dendrimer (G4-SA) was evaluated as a means of preventing adhesion of 3 influenza A subtypes (H1N1, H2N2, and H3N2). In hemagglutination-inhibition assays, G4-SA was found to inhibit all H3N2 and 3 of 5 H1N1 influenza subtype strains at concentrations 32-170 times lower than those of SA monomers. In contrast, G4-SA had no ability to inhibit hemagglutination with H2N2 subtypes or 2 of 5 H1N1 subtype strains. In vivo experiments showed that G4-SA completely prevented infection by a H3N2 subtype in a murine influenza pneumonitis model but was not effective in preventing pneumonitis caused by an H2N2 subtype. Polyvalent binding inhibitors have potential as antiviral therapeutics, but issues related to strain specificity must be resolved.

Development of immune response that protects mice from viral pneumonitis after a single intranasal immunization with influenza A virus and nanoemulsion.

Nanoemulsion, a water-in-oil formulation stabilized by small amounts of surfactant, is non-toxic to mucous membranes and produces biocidal activity against enveloped viruses. We evaluated nanoemulsion as an adjuvant for mucosal influenza vaccines. Mice (C3H/HeNHsd strain) were vaccinated intranasally with 5 x 10(5) plaque forming units (pfu) of influenza A virus (Ann Arbor/6/60 strain) and a nanoemulsion mixture. The mice were challenged on day 21 after immunization with an intranasal lethal dose of 2 x 10(5) pfu of virus. Animals vaccinated with the influenza A/nanoemulsion mixture were completely protected against infection, while animals vaccinated with either formaldehyde-killed virus or nanoemulsion alone developed viral pneumonitis and died by day 6 after the challenge. Mice vaccinated with virus/nanoemulsion mixture had rapid cytokine responses followed by high levels of specific anti-influenza immunoglobulin G (IgG) and immunoglobulin A (IgA) antibodies. Specificity of the immune response was confirmed by assessment of the proliferation and cytokine production in splenocytes. This paper demonstrates that nanoemulsion can be employed as a non-toxic mucosal adjuvant for influenza virus vaccine.

A novel inactivated intranasal respiratory syncytial virus vaccine promotes viral clearance without TH2 associated Vaccine-Enhanced disease

Background Respiratory syncytial virus (RSV) is a leading cause of bronchiolitis and pneumonia in young children worldwide, and no vaccine is currently available. Inactivated RSV vaccines tested in the 1960s led to vaccine-enhanced disease upon viral challenge, which has undermined RSV vaccine development. RSV infection is increasingly being recognized as an important pathogen in the elderly, as well as other individuals with compromised pulmonary immunity. A safe and effective inactivated RSV vaccine would be of tremendous therapeutic benefit to many of these populations. Principal Findings In these preclinical studies, a mouse model was utilized to assess the efficacy of a novel, nanoemulsion-adjuvanted, inactivated mucosal RSV vaccine. Our results demonstrate that NE-RSV immunization induced durable, RSV-specific humoral responses, both systemically and in the lungs. Vaccinated mice exhibited increased protection against subsequent live viral challenge, which was associated with an enhanced Th1/Th17 response. In these studies, NE-RSV vaccinated mice displayed no evidence of Th2 mediated immunopotentiation, as has been previously described for other inactivated RSV vaccines. Conclusions These studies indicate that nanoemulsion-based inactivated RSV vaccination can augment viral-specific immunity, decrease mucus production and increase viral clearance, without evidence of Th2 immune mediated pathology.

A rapid staining technique for the detection of the initiation of germination of bacterial spores

Aims: We propose to apply the Wirtz‐Conklin staining technique to evaluate spore germination.

The fungicidal activity of novel nanoemulsion (X8W60PC) against clinically important yeast and filamentous fungi.

Surfactant nanoemulsions are water in oil preparations that proved to have a broad spectrum biocidal activity against a variety of microorganisms including Gram-positive and Gram-negative bacteria, spores and enveloped viruses. These preparations are non-toxic to the skin, mucous membrane and gastrointestinal tissues at biocidal concentrations. In this study, 0.1% of the nanoemulsion designated X8W60PC has shown fungicidal activity against yeast including Candida albicans and C. tropicalis in 15 minutes. C. tropicalis was more sensitive than C. albicans, which required a longer time or a higher concentration of the nanoemulsion to achieve killing. Neutral to slightly alkaline pH was more effective in killing the yeast cells than acidic pH. Using the minimum inhibitory concentration assay, 0.08% of the nanoemulsion was inhibitory to C. albicans, and parapsilosis and filamentous fungi including Microsporum gypseum,Trichophyton mentagrophytes,Trichophyton rubrum,Aspergillus fumigatus andFusarium oxysporum.None of the individual ingredients was as effective a fungicidal as the nanoemulsion at equivalent concentration. This shows that the nanoemulsion structure is an important factor in the anti-fungal activity. The X8W60PC has great potential as a topical anti-fungal agent and further investigation into the mechanism of fungicidal action is warranted.

Topical nanoemulsion therapy reduces bacterial wound infection and inflammation after burn injury

BACKGROUND Nanoemulsions are broadly antimicrobial oil-in-water emulsions containing nanometer-sized droplets stabilized with surfactants. We hypothesize that topical application of a nanoemulsion compound (NB-201) can attenuate burn wound infection. In addition to reducing infection, nanoemulsion therapy may modulate dermal inflammatory signaling and thereby lessen inflammation following thermal injury. METHODS Male Sprague-Dawley rats underwent a 20% total body surface area scald burn to create a partial-thickness burn injury. Animals were resuscitated with Ringers lactate solution and the wound covered with an occlusive dressing. At 8 hours after injury, the burn wound was inoculated with 1 x 10(6) colony-forming units (CFUs) of Pseudomonas aeruginosa. NB-201, NB-201 placebo, 5% mafenide acetate solution, or 0.9% saline (control) was applied onto the wound at 16 and 24 hours after burn injury. Skin was harvested 32 hours postburn for quantitative wound culture and determination of inflammatory mediators in tissue homogenates. RESULTS NB-201 decreased mean bacterial growth in the burn wound by 1,000-fold, with only 13% (3/23) of animals having P. aeruginosa counts greater than 10(5) CFU/g tissue versus 91% (29/32) in the control group (P < .0001). Treatment with NB-201 attenuated neutrophil sequestration in the treatment group as measured by myeloperoxidase assay and by histology. It also significantly decreased levels of proinflammatory cytokines (interleukin [IL]-1beta and IL-6) and the degree of hair follicle cell apoptosis in skin compared to saline-treated controls. CONCLUSION Topical NB-201 substantially decreased bacterial growth in a partial-thickness burn model. This decrease in the level of wound infection was associated with an attenuation of the local dermal inflammatory response and diminished neutrophil sequestration. NB-201 represents a novel potent antimicrobial and anti-inflammatory treatment for use in burn wounds.

Safety and immunogenicity of a novel nanoemulsion mucosal adjuvant W805EC combined with approved seasonal influenza antigens.

BACKGROUND Improving the systemic and mucosal immune response following intranasal vaccination could enhance disease protection against respiratory pathogens. We assessed the safety and immunogenicity of a novel nanoemulsion mucosal adjuvant W(80)5EC combined with approved seasonal influenza antigens. METHODS This was a first-in-human Phase I study in 199 healthy adult volunteers randomized to receive a single intranasal administration of 5%, 10%, 15% or 20% W(80)5EC, combined with 4 or 10 μg strain-specific Fluzone(®) HA, compared with intranasal PBS, intranasal Fluzone(®), or 15 ug strain-specific intramuscular Fluzone(®). Safety was evaluated by physical examination, laboratory parameters, symptom diaries, and adverse event reports. Serum HAI titers and nasal wash IgA were assessed at baseline as well as 28 and 60 days after vaccination. RESULTS W(80)5EC adjuvant combined with seasonal influenza antigens was well tolerated without safety concerns or significant adverse events. The highest dose of 20% W(80)5EC combined with 10 μg strain-specific HA elicited clinically meaningful systemic immunity based on increases in serum HAI GMT and ≥ 70% seroprotection for all 3 influenza strains, as well as a rise in antigen-specific IgA in nasal wash specimens. CONCLUSIONS W(80)5EC adjuvant was safe and well tolerated in healthy adult volunteers and elicited both systemic and mucosal immunity following a single intranasal vaccination.