Hiromi Toyoizumi

Enhanced sensitisation of mice with diphtheria tetanus acellular pertussis vaccine to local swelling reaction to the booster immunisation

Severe local swelling has been regarded as a serious safety problem for the booster immunisations of diphtheria tetanus acellular pertussis combined (DTaP) vaccine and DT combined toxoids (DT-td). We attempted to search for the factor of DTaP vaccines possibly contributing to the enhanced local reaction by using the mouse hind paw swelling reaction. Mice were immunised intramuscularly with DTaP vaccine twice at 1-month interval and were challenged their hind paw with one of the antigens of DTaP vaccine 2 weeks later. D-td was shown to elicit the strongest swelling among the vaccine antigens. No causal relationship was found between the swelling and the level of immunoglobulin G (IgG) or IgE in mice. Residual pertussis toxin (PT) activity of DTaP vaccines for immunisation was shown to play a role in the enhanced sensitisation of mice to the D-td-related hind paw swelling.

A Limulus Amoebocyte Lysate Activating Activity (LAL Activity) That Lacks Biological Activities of Endotoxin Found in Biological Products

Pyrogenic substances in influenza HA (IHA) vaccine have been controlled by the pyrogen test or the mouse body weight decreasing toxicity (BWD) test. We examined the possibility of replacing the animal tests with the endotoxin test. Commercial IHA vaccines were found to show considerable levels of LAL activity ranging from 0.2 to 160 EU/ml. However, a batch of the vaccine having even 100 EU/ml of LAL activity showed neither pyrogenicity in rabbits nor tumor necrosis factor alpha (TNF‐α) induction in RAW264.7 cells. The LAL activity of IHA vaccine was abolished by a monoclonal antibody that recognizes LPS‐binding epitope of LAL factor C. The activity of IHA vaccine showed different physicochemical properties from those of LAL activity of endotoxin. LAL activity of endotoxin is known to be sensitive to polymyxin B treatment and was found to be resistant to polyoxyethylene 10 cetyl ether (Brij56) treatment. On the contrary, the LAL activity of IHA vaccine was shown to be resistant to polymyxin B but sensitive to Brij56 treatment. The difference in sensitivity of the two LAL activities to polymyxin B and Brij56 might suggest the possibility of their discriminative measurements.

Evaluation of endotoxin content of diphtheria–tetanus-acellular pertussis combined (DTaP) vaccines that interfere with the bacterial endotoxin test

Applicability of the endotoxin test to diphtheria-tetanus-acellular pertussis combined (DTaP) vaccines was examined. We found some DTaP vaccines that strongly interfered with Limulus amoebocyte lysate (LAL) activity of endotoxin without affecting lethal activity of endotoxin in D-galactosamine-treated mice. LAL activity that was interfered in such vaccines increased apparently after the treatment with phosphate buffer at 4 degrees C for a week. The DTaP vaccines that interfered with the endotoxin test showed no significant effect on endotoxin activity in inducing tumor necrosis factor-alpha (TNF-alpha) in rabbit peripheral blood. The in vitro TNF-alpha induction assay was, therefore, suggested to be an appropriate assay method for the quantitative detection of the endotoxin activity in DTaP vaccines.

A quantitative in vitro assay method for detecting biological activity of endotoxin using prostaglandin E2 induction in rabbit peripheral blood.

The pyrogen test and the endotoxin test (the LAL test) have been playing crucial roles in detecting endotoxin in parenteral drugs. The current test methods, however, have disadvantages such as requiring a large number of animals or an inadequacy in evaluation of in vivo endotoxin activity. We attempted to establish a new assay method that can overcome the shortcomings of the current methods. We standardized the in vitro assay method by the use of prostaglandin E2 (PGE2) induction from peripheral blood of rabbits for detecting endotoxin activity. A linear dose‐response regression was attained from approximately 0.15 to 5 endotoxin units/ml of Japanese national reference standard endotoxin by the in vitro assay. The assay showed a fine correlation with the pyrogen test but not with the LAL test, when endotoxins from various bacterial sources were tested. The in vitro assay was also shown to have the capability of detecting a synergistic effect of endotoxin and parenteral drugs. The in vitro PGE2 induction test using rabbit blood was, therefore, suggested to be the appropriate test method for guaranteeing the same level of safety of parenteral drugs as the pyrogen test does.

Augmenting effect of antibiotics on endotoxin activity may cause a safety problem.

Enhancing/interfering effect of antibiotics on endotoxin was evaluated using the endotoxin test and the cell line assay in 28SC cells that has a responsiveness consistent with that of human peripheral blood. When a total of 21 products of seven different kinds of antibiotics were tested, none showed any significant effect on the endotoxin test at its therapeutic dose. However, aminoglycosides showed a significant augmenting effect on IL‐6 induction of endotoxin in 28SC cells. Detailed examination of the augmenting effect was made on spectinomycin in the in vitro cell line assay and also in the lethal endotoxin challenge assay in D‐galactosamine‐treated mice. Spectinomycin also enhanced the endotoxin lethality in D‐galactosamine‐treated mice. A kinetic analysis in endotoxin‐sensitized 28SC cells revealed that the augmentation takes place as quickly as 10 min after spectinomycin treatment. Accordingly, a special caution concerning the augmenting effect was assumed necessary for the safety control of antibiotic products as well as for selecting antibiotics for the therapeutic use.