Acthib

Mice were anesthetized with isoflurane (FUJIFILM Wako Pure Chemical, Osaka, Japan) and then subcutaneously immunized with a total volume of 200 μl PBS containing either 0.01 μg of the Hib capsular polysaccharide PRP, 0.01 μg of PRP plus 0.024 μg of tetanus toxoid (TT) (EMD Millipore, Burlington, MA, United States), Haemophilus B PRP–TT conjugate vaccine (ActHIB; Sanofi, Tokyo, Japan) equivalent to 0.01 μg of PRP or 1 μg of PRP with or without 1 μg of Alcaligenes lipid A (Wang et al., 2020 (link)), or PBS only. Mice received three immunizations at 1-week intervals. One week after the final immunization, blood was harvested from the mice and kept on ice until centrifuged at 4°C, 3,000 × g for 10 min. The serum was transferred into a fresh tube and stored at −80°C.

Each 0.5ml dose of Prevenar13^® (Wyeth/Pfizer) contains two micrograms of Pneumococcal polysaccharide serotypes 1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 23F and four micrograms of pneumococcal polysaccharide serotype 6B. It also contains sodium chloride and water for injection. The control agent, Hib (ActHIB^®- Sanofi Pasteur) vaccine consists of the Haemophilus influenzae type b capsular polysaccharide (polyribosyl-ribitol-phosphate, PRP), covalently bound to tetanus toxoid. Both the lyophilised ActHIB vaccine powder and saline diluent contain no preservative. When reconstituted with saline, each single dose of 0.5 mL is formulated to contain 10 mcg of purified capsular polysaccharide conjugated to 24 mcg of inactivated tetanus toxoid, and 8.5% of sucrose. Each vaccine was administered by intramuscular injection provided in single-dose syringes. The vaccines were commercially labelled.
All vaccinations were administered according to specific product guidelines by a qualified immunisation nurse. The vaccination details were recorded on the participant’s clinic card as well as participant’s study CRF. As the clinic card remains stored at the Diana Centre, there was a backup to ensure that all children get both types of the vaccines at an appropriate schedule. Parents and guardians were shielded from seeing which vaccine was given.

DTaP-mIPV and DTaP-vIPV contain inactivated poliovirus (IPV) from MRC-5 and Vero cell sources, respectively. The production-scale lots of DTaP-mIPV and DTaP-vIPV were manufactured by Sanofi Limited, Toronto, ON, Canada. Both vaccines are formulated with 6 μg/mL of PRN and contain the same amounts of pertussis toxoid, filamentous hemagglutinin, fimbriae types 2 and 3, diphtheria toxoid, tetanus toxoid, and aluminum phosphate adjuvant. The production-scale lots of lyophilized Act-Hib (Haemophilus b conjugate vaccine) were manufactured by Sanofi, Marcy L’Étoile, France. To generate DTaP-IPV/Hib vaccine, DTaP-IPV vaccine was used to reconstitute the lyophilized Act-Hib as per product monograph.
For the heat stress studies, DTaP-vIPV was heated to 60 °C for 1, 2, 4, 7, 14, 21, and 28 days prior to testing.
For use in the assay validation studies, lab-scale mock formulations of DTaP-vIPV were prepared containing the same antigen content, buffer components, and AlPO₄ adjuvant as the production-scale vaccine, with the exception of PRN. PRN (adjuvanted with AlPO₄) was then spiked into the mock DTaP-vIPV to generate vaccines with varying levels of PRN antigen.