M. R. W. Brown

Effectiveness of liposomes as adjuvants of orally and nasally administered tetanus toxoid

Abstract Tetanus toxoid was incorporated into liposomes of equimolar concentrations of distearoyl phosphadylcholine (DSPC) and cholesterol. We investigated the non-parenteral delivery of free or liposome entrapped tetanus toxoid to guinea pigs and measured the subsequent IgG anti-tetanus-antibody response, using an ELISA. Liposome formulation significantly improved the immune response when compared to the free antigen when delivered via the nasal, oral and i.m. routes. However, 10 × concentration of tetanus toxoid entrapped in DSPC introduced mucosally (nasally and orally) was necessary to produce an IgG antibody titre similar to those obtained via the i.m. delivery. This study suggests that liposomes, administered through the oral and nasal routes, have considerable potential as mucosal adjuvants and warrant further investigation.

Growth and sporulation characteristics of Bacillus megaterium under different conditions of nutrient limitation.

The quantitative nutritional requirements to achieve specific cell densities have been studied for B. megaterium. Growth of batch cultures was separately limited by depletion of glucose, ammonium, sulphate, potassium, phosphate, manganese and magnesium. Maximum population density (E420) for graded concentrations of each limiting nutrient was plotted against nutrient concentration and a linear plot was obtained below a critical concentration. Under conditions of magnesium depletion, two phases of growth occurred separated by a plateau. Proposals are made for the use of these cultures in drug resistance studies. Sporulation occurred in all cultures except those limited by potassium, manganese or magnesium. Spores were produced in magnesium‐limited cultures provided that glucose was simultaneously depleted. Spores produced under different conditions of nutrient depletion varied in germination characteristics, heat resistance and spore volume.

Evaluation of glycine as an inactivator of glutaraldehyde

Glycine was evaluated as an inactivator of the sporicidal activity of glutaraldehyde. Spores from glucose depleted cultures of Bacillus stearothermophilus grown in a chemically defined medium were used. When glycine is used as an inactivator of glutaraldehyde, it lowers the pH value of the solution. Glycine 1% failed to inactivate 0.5% or higher concentrations of alkaline glutaraldehyde in sporicidal studies. If viable counts cannot be performed within the first hour after inactivation, the concentration of glycine should be at least 2% to inactivate effectively 2% alkaline glutaraldehyde.

Increasing the probability of sterility of medicinal products

Calculations show that official tests for sterility offer unacceptably low degrees of assurance of sterility. Some batches of heat treated articles with 10% contamination can pass the E.P. test on about 92% of occasions. It is proposed that viable counts be made on products immediately before the terminal inactivation procedure and that upper limits be set for the level of contamination. Samples of the product should be inoculated with spores of known resistance characteristics and also with samples of swabs from the production area. Such inoculated products should then be tested for absence of viable organisms, after being subjected to the terminal inactivation procedure. This test should be coupled with close environmental and process control and personnel education. It is recommended that these procedures replace the conventional test for sterility. The use of spores as direct and independent indicators of sterility, especially where the lethal conditions cannot be monitored instrumentally is recommended. A flexible approach is proposed for the use of the ‘lethality factor’ suitable for a terminal heat inactivation procedure; this would depend on the nature of the product and the standard of monitoring facilities and personnel. A change in the language is proposed. A medicinal product processed such that an acceptable probability of sterility exists, should be designated not as sterile but as safe.

Mucosal delivery of herpes simplex virus vaccine.

The mucosal route for the production of mucosal and systemic herpes simplex virus (HSV) antibodies was investigated using HSV1 subunit vaccine administered to guinea pigs. Groups of test animals (n = 13) were dosed, nasally or vaginally and compared with those injected subcutaneously (s.c.). The vaccines, in aqueous or gel form, were administered 5 and 3 weeks prior to vaginal challenge with HSV2 suspension. Control infected and non-infected animals were included for comparison. Animals which were vaccinated s.c. were shown to respond to subsequent infection with HSV by the production of serum HSV-specific IgG (and IgA) but negligible amounts of vaginal IgG and IgA. Control non-infected and infected-only groups produced none and only a small amount of vaginal HSV-specific antibodies, respectively. Substantial protection against HSV2 infection of the female guinea pig genital tract was provided by s.c. immunization with HSV vaccine. Protection was evaluated in terms of the reduction of histopathological lesions and clinical signs in vaccinated and control animals. The serum humoral response to nasal delivery in phosphate-buffered saline was comparable, and was superior for vaginal washes to that of parenteral vaccination. The nasally delivered free antigen gave significant (p < or = 0.05) reduction in the severity of the disease and higher levels of specific serum and vaginal immunoglobulin antibodies to HSV when compared with non-immunized infected-only controls, probably due to uptake of antigenically intact protein. Vaginal gel treatment slightly reduced the severity of the illness and gave higher humoral responses than those induced by vaginally delivered free antigen. Findings also indicate that these mucosal immune responses were produced at a site distant from the site of vaccination, suggesting a common immunological system.

Release of dipicolinic acid and calcium and activation of Bacillus stearothermophilus spores as a function of time, temperature and pH

The kinetics of release of dipicolinic acid and calcium from Bacillus stearothermophilus spores and the rate of increase of colony count have been shown to be determined by conditions of time, temperature and pH. The apparent activation energies for release of dipicolinic acid, calcium and colony count increase were similar. The results support the hypothesis that breaking of dormancy involves a rupture of dipicolinic acid bonds and that the nature of these bonds rather than the dipicolinic acid content determines dormancy and resistance.

Additivity of action between polysorbate 80 and polymyxin B towards spheroplasts of Pseudomonas aeruginosa NCTC 6750

When polymyxin B and polysorbate 80 were used together against spheroplasts of Pseudomonas aeruginosa, the activities were found to be additive. These substances have previously been reported to act synergistically against P. aeruginosa, but little or no intrinsic activity towards intact cells has been attributed to polysorbate 80. We suggest that in addition to enhancing polymyxin B penetration to the cytoplasmic membrane, polysorbate 80 may also act as an antimicrobial agent when polymyxin‐induced damage to the outer membrane facilitates the surfactants passage through the cell envelope.

The Immune Responses and Protective Efficacy of the Skinner Herpes Simplex Virus Vaccine Administered by Oral and Nasal Routes in Liposomes

AbstractThe serum and mucosal response in female guinea pigs vaccinated non-parenterally with the Skinner Herpes Simplex virus (HSV) vaccine, was investigated. We compared the abilities of liposome – incorporated vaccine delivered orally/nasally in inducing an immune response and reducing the severity of herpes infection to the subcutaneously delivered antigen. Our results show that nasal delivery for the vaccine entrapped in distearoylphosphatidylcholine (DSPC) liposomes resulted in a significant (P< 0.05) increase in levels of serum antibodies and also provided a substantial (P< 0.01) protection against intravaginal HSV infection compared to non-immunized infected-only animals. Liposome – incorporated antigen when orally administered only moderately increased serum antibody levels and gave no significant remission of the disease when compared to the infected only group. The nasal delivery gave similar results to those achieved via the parenteral route. Apparently no specific sIgA to HSV was stimulated i...