Mice
During the animal research period, the mouse were housed in filter-top cages under semi-specific pathogenfree conditions and food and water are available freely. All animal research procedures were performed in accordance with the Laboratory Animals Welfare Act, the Guide for the Care and Use of Laboratory Animals and the Guidelines and Policies for Rodent Experiments provided by the IACUC (Institutional Animal Care and Use Committee) in the School of Medicine, The Catholic University of Korea. (Approval number: CUMS-2019-0100-01).
Immunization
Four-week-old BALB/c female mice from Orient-bio Co., Ltd. (Seongnam, Korea) were used. The mice were divided into 4 groups (30 mice per group) according to booster vaccine types: negative control boosted with saline, positive control with licensed Tdap vaccine (BoostrixTM) from GSK (GlaxoSmithKline, Rixensart, Belgium) and two study groups; one study group with the previous GC Pharma Tdap vaccine GC3111 and the other study group with the enhanced FHA antigen of GC 3111 vaccine. All mice were vaccinated with one-fourth of the human dose (0.125 mL) via intramuscular (quadriceps muscle) injection and immunized with two doses of primary diphtheria-tetanus-acellular pertussis (DTaP) vaccine (provided by GC Pharma) at 3-week intervals, except for the negative control group, which was vaccinated with saline before booster vaccination. The study was conducted according to previous murine model studies at our laboratory at the Vaccine Bio Research Institute [11-12]. All Tdap vaccine components were equivalent to PT 8 μg, FHA 8 μg, and PRN 2.5 μg. The vaccination and assay schedule is described in Fig. 1.
Humoral immunity assessment
Blood samples from the retro-bulbar venous plexuses were collected in each group at 1 week before booster vaccination (n=6 per group) and 2, 4 week after booster vaccination (n=10). When sampling the blood, all mice were anesthetized with tiletamine, zolazepam and xylazine via intra peritoneal injection except last sampling. At 4 week after booster vaccination, mice were euthanized by 2% isoflurane inhalation while sampling and sacrificed via CO2 inhalation. The humoral immunogenicity against pertussis antigens (anti-PT IgG, anti-FHA IgG and anti-PRN IgG) was evaluated by commercially available ELISA kits (Alpha Diagnostic International Inc., San Antonio, TX, USA). Additionally, anti-diphtheria toxoid (DT) IgG and antitetanus toxoid (TT) IgG titres were measured using commercially available ELISA kits (Alpha Diagnostic International Inc. San Antonio, TX, USA). All final results were analysed through optimal density using an Epoch ELISA plate reader (BioTek Instrumetns Inc., Winooski, VT, USA). Antibody titres of each tested antigen were compared between groups at each time point.
Cellular mediated immunity assessment
Four weeks after the booster vaccination, mouse spleen cells (n=5 per group) were prepared in RPMI-1640 (HyClone, GE Healthcare Life Sciences, SouthLogan, Utah, USA) medium containing penicillin, streptomycin, and 10% FBS. For cell-based experiments, 1 μg/mL pokeweed mitogen (PWM; Sigma-Aldrich, St. Louis, MO, USA) was used as a positive control, and the following three stimulators were tested: 1 × 106 CFU/mL of heat inactivated Bordetella pertussis (hBp), PT (8 μg/mL), FHA (8 μg/mL) and PRN (4 μg/mL) vaccine antigens, and the mixture of the two (hBp + antigens). Splenocytes (5 × 106 cells/mL) were treated with three simulators separately and cultured in 6-well plates for 3 days. Subsequently, the cytokine response was assessed by analysing the supernatant using ELISA kits (R&D Systems, Minneapolis, MN, USA).
Intranasal challenge test
The protective efficacy against B. pertussis infection was assessed with intranasal clearance tests. The challenge B. pertussis strain obtained from a Korean adult pertussis patient was supplied from the Korean Centers for Disease Control & Prevention (No. 13674) and was intranasally inoculated at 6 × 106 colony forming units (CFUs) 4 weeks after booster vaccination. Four mice in each group were euthanized by 2% isoflurane inhalation and their lungs were extracted 2 h, 2 days, 5 days and 8 days after infection. The extracted lungs were homogenized in 10 mL of PBS and diluted to concentrations of 10−1, 10−3 and 10−5. Each diluted homogenate was incubated on Bordet-Gengou agar media at 37 °C for 5 days. CFUs on each media were determined, and mean CFUs were compared between groups at each time point.
Statistical analysis
All results are expressed as the means ± standard errors of the means (SEM) and compared by two-way ANOVA with Tukey’s multiple comparison test. Statistical analysis was performed using GraphPad PrismTM software v7.02 (GraphPad, San Diego, CA, USA), and statistical significance was defined as a p value (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).