Collaborative study for the calibration of the replacement International Standard for pertussis toxin for use in histamine sensitisation and CHO cell clustering assays.

Pertussis toxin (PT) in its detoxified form is one of the major protective antigens in vaccines against Bordetella pertussis (whooping cough). Reference preparations of native PT are required for the quality control of pertussis vaccines. Stocks of the first WHO International Standard (IS) for PT (JNIH-5) were low and a replacement was required. One candidate material was donated by a vaccine manufacturer to NIBSC. It was formulated, lyophilised into sealed glass ampoules and coded 15/126. An international collaborative study assessed the suitability of this material to replace JNIH-5. Fourteen laboratories from 12 countries took part in the study. Eleven laboratories performed lethal murine histamine sensitisation assay (HIST), 14 performed Chinese Hamster Ovary (CHO) cell clustering assay. International Units (IU) were assigned to the material using these assays as they were used to assign units to JNIH-5. It was found that, unlike JNIH-5, the activities of 15/126 in HIST and CHO cell assays did not agree and therefore different unitage for each assay was assigned. The preparation 15/126 was established as the Second WHO IS for PT for HIST and CHO cell assays. It was assigned a unitage of 1,881 IU/ampoule in HIST and 680 IU/ampoule in the CHO cell clustering assay.

An in vitro assay system as a potential replacement for the histamine sensitisation test for acellular pertussis based combination vaccines.

The histamine sensitisation test (HIST) for pertussis toxin is currently an official batch release test for acellular pertussis containing combination vaccines in Europe and North America. However, HIST, being a lethal endpoint assay, often leads to repeated tests due to large variations in test performance. Although a more precise HIST test based on measurement of temperature reduction after the histamine challenge is used in Asian countries, this test still uses animals. An in vitro test system based on a combination of enzyme coupled-HPLC and carbohydrate-binding assays with results analysed by a mathematical formula showed a good agreement with the in vivo HIST results based on measurement of temperature reduction after histamine challenge. The new in vitro test system was shown to be a potential alternative to the current in vivo HIST.

Immune interaction between components of acellular pertussis-diphtheria-tetanus (DTaP) vaccine and Haemophilus influenzae b (Hib) conjugate vaccine in a rat model.

We have previously shown that, consistent with clinical trial results, the immune response to a Haemophilus influenzae b (Hib) conjugate vaccine in a rat model was compromised and modulated when given combined with a DTaP3 vaccine, as compared to both vaccines given separately. The present study extended our investigation to evaluate the immunogenicity of all DTaP3 components in combined versus separate administration of Hib with DTaP3 and investigated immune interactions between Hib and individual components of DTaP3. Rats were immunised with Hib and DTaP3 or with Hib and individual DTaP3 components. Cellular and humoral immune responses to Hib and DTaP3 components were evaluated. Our results indicate that the immunogenicity of DTaP3 components was similar or greater in combined versus separate administration of Hib and DTaP3. Moreover, combined administration of Hib and TT reduced immunogenicity of both Hib and TT. Hib immunogenicity was also significantly reduced when given combined with FHA and following adsorption to Al(OH)3.