Immunogenicity of a new enhanced tetanus-reduced dose diphtheria-acellular pertussis (Tdap) vaccine against Bordetella pertussis in a murine model.

BACKGROUND: The necessity of the tetanus-reduced dose diphtheria-acellular pertussis (Tdap) vaccine in adolescence and adults has been emphasized since the resurgence of small-scale pertussis in Korea and worldwide due to the waning effect of the vaccine and variant pathogenic stains in the late 1990s. GreenCross Pharma (GC Pharma), a Korean company, developed the Tdap vaccine GC3111 in 2010. Recently, they enhanced the vaccine, GC3111, produced previously in 2010 to reinforce the antibody response against filamentous hemagglutinin (FHA). In this study, immunogenicity and efficacy of the enhanced Tdap vaccine compared and evaluated with two Tdap vaccines, GC3111 vaccine produced in 2010 previously and commercially available Tdap vaccine in a murine model. METHODS: Two tests groups and positive control group of Balb/c mice were primed with two doses of the diphtheria-tetanus-acellular pertussis (DTaP) vaccine followed by a single booster Tdap vaccine at 9 week using the commercially available Tdap vaccine or 2 Tdap vaccines from GC Pharma (GC3111, enhanced GC3111). Humoral response was assessed 1 week before and 2 and 4 weeks after Tdap booster vaccination. The enhanced GC3111 generated similar humoral response compare to the commercial vaccine for filamentous hemagglutinin (FHA). The interferon gamma (IFN-γ) (Th1), interleukin 5 (IL-5) (Th2) and interleukin 17 (IL-17) (Th17) cytokines were assessed 4 weeks after booster vaccination by stimulation with three simulators: heat inactivated Bordetella pertussis (hBp), vaccine antigens, and hBp mixed with antigens (hBp + antigen). A bacterial challenge test was performed 4 weeks after booster vaccination. RESULTS: Regarding cell-mediated immunity, cytokine secretion differed among the three simulators. However, no difference was found between two test groups and positive control group. All the vaccinated groups indicated a Th1 or Th1/Th2 response. On Day 5 post-bacterial challenge, B. pertussis colonies were absent in the lungs in two test groups and positive control group. CONCLUSIONS: Our results confirmed the immunogenicity of GC Pharma's Tdap vaccine; enhanced GC3111 was equivalent to the presently used commercial vaccine in terms of humoral response as well as cell-mediated cytokine expression.

Assessment of safety and efficacy against Bordetella pertussis of a new tetanus-reduced dose diphtheria-acellular pertussis vaccine in a murine model.

BACKGROUND: Tetanus-reduced dose diphtheria-acellular pertussis (Tdap) vaccination during adolescence was introduced in response to the resurgence of pertussis in various countries. A new Tdap vaccine was manufactured in Korea as a countermeasure against a predicted Tdap vaccine shortage. This study was performed to evaluate the immunogenicity, safety, and protection efficacy against Bordetella pertussis of the new Tdap vaccine in a murine model. METHODS: Four-week-old BABL/c mice were used for assessment of immunogenicity and protection efficacy. A single dose of primary diphtheria-tetanus-acellular pertussis (DTaP) vaccine was administered, followed by a single dose of Tdap booster vaccine after a 12-week interval. Anti-pertussis toxin (PT), anti-filamentous hemagglutinin (FHA), and anti-pertactin (PRN) IgG titers were measured before primary vaccination, and before and after booster vaccination. An intranasal challenge test was performed after booster vaccination to determine protection efficacy. To assess safety, mouse weight gain test and leukocytosis promotion test were performed using 4-week-old ddY female mice. RESULTS: Anti-PT and anti-FHA IgG titers after booster vaccination were significantly higher than those before booster vaccination with either the new vaccine or a commercially available Tdap vaccine (P = 0.01 for all occasions). After booster vaccination, no significant difference was observed between the two vaccines in antibody titers against pertussis antigens (P = 0.53 for anti-PT IgG, P = 0.91 for anti-FHA IgG, P = 0.39 for anti-PRN IgG). In the intranasal challenge test, inoculated B. pertussis was eradicated 7 days after infection. On days 4 and 7 after infection, colony counts of B. pertussis were not significantly different between the new and positive control vaccine groups (P = 1.00). Mean body weight changes and leukocyte counts of the new vaccine, positive control, and negative control groups were not significantly different 7 days after vaccination (P = 0.87 and P = 0.37, respectively). All leukocyte counts in the new vaccine group were within a mean ± 3 standard deviations range. CONCLUSIONS: A murine model involving a single dose primary DTaP vaccination followed by a single dose Tdap booster vaccination can be used for non-clinical studies of Tdap vaccines. The new Tdap vaccine manufactured in Korea exhibited comparable immunogenicity, protection efficacy, and safety with a commercially available Tdap vaccine.

Development and implementation of standardized method for detecting immunogenicity of acellular pertussis vaccines in Korea.

PURPOSE: There is no standard method for confirming the immunogenicity of acellular pertussis vaccines. We tried to develop a local standard method for evaluating the immunogenicity of the three-component of acellular pertussis vaccines which was developed by a Korean local company. MATERIALS AND METHODS: The developed pertussis antigens (pertussis toxin, filamentous hemagglutinin, pertactin) were evaluated by in-house enzyme-linked immunosorbent assay (ELISA) using 189 negative sera, 25 positive sera, and 73 paired sera (pre- and post-Tdap [tetanus, diphtheria, and acellular pertussis] vaccinated sera). ELISA units were calculated by the reference line method, compared with World Health Organization reference sera, and the cut-off value was calculated using negative sera. RESULTS: When compared to National Institute for Biological Standards and Control control antigen (NIBSC) control antigens, the developed pertussis toxin (PT) and filamentous haemagglutinin (FHA) antigens were 203.48 and 61.60 IU/µg, respectively. Each in-house ELISA was established by validating the coefficients of variation % (PT, 11.53%; FHA, 8.60%; pertactin [PRN], 9.86%) obtained from the results of inter- and intra-assay variation. Also, the cut-off values of PT, FHA, and PRN were 11.65, 38.95, and 5.66 EU/mL, respectively. The distributions of antibody levels in paired showed that 93.15% (68/73) in anti-PT IgG, 97.26% (72/73) in anti-FHA IgG, and 100% in anti-PRN IgG were higher than a 100% increase after vaccination. Additionally, the values of 89.04% (65/73) in anti-PT IgG, 97.26% (72/73) in anti-FHA IgG, and 100% in anti-PRN IgG were below each cut-off point. CONCLUSION: We established an in-house ELISA method using self-developed antigens, and these immunoassays have provided a way to standardize measuring the immunogenicity of newly developed vaccines, through single- and dual-serology.

Enzyme-linked immunosorbent assay for detecting anti-pertussis toxin antibody in mouse.

PURPOSE: Although the DTaP and Tdap vaccines used to prevent pertussis have been used for a long time, there is no standard method for measuring pertussis antigens. Therefore, this preliminary study was conducted to develop an enzyme-linked immunosorbent assay method using an animal model for measuring antibodies against pertussis toxin, the most important pertussis pathogenic antigen, in the sera of vaccinated mice. MATERIALS AND METHODS: Bordetella pertussis Tohama phase I was cultured for 24-30 hours, and then pertussis toxin was purified from the culture medium by chromatography. Purified pertussis toxin was diluted in phosphate-buffered saline-coating buffer, and 100 µL of diluted pertussis toxin was added to each well and reacted at room temperature for 4 hours. Positive serum was diluted to 1/1,250-1/80,000 and negative serum was diluted to 1/50 to determine the coating concentration with the optimal signal/noise ratio. Optimal test conditions were confirmed from the dilution factors of the secondary antibody and streptavidin horseradish peroxidase (SA-HRP). RESULTS: Optimal conditions were as follows: 4 µg/mL for coating antigen; 1/40,000 for secondary antibody; and 1/1,000 for the SA-HRP dilution factor. Comparison of the sera obtained from mice treated with a developing vaccine and commercial vaccine with National Institute for Biological Standard and Control standard serum under the established conditions showed the following results: 1,300.62, 534.94, and 34.85, respectively. CONCLUSION: The method developed in this study is suitable for measuring anti-pertussis toxin antibodies and may be applicable for clinical sample analysis or indirect diagnosis of pertussis.

Immunogenicity and protective efficacy of a newly developed tri-component diphtheria, tetanus, and acellular pertussis vaccine in a murine model.

BACKGROUND/PURPOSE: Although assessing the immunogenicity and protective efficacy of acellular pertussis (aP) vaccines via murine model studies faces limitations, preliminary assessments have been achieved by evaluating respiratory challenge and humoral and cellular immunity. METHODS: We performed a long-term intranasal respiratory challenge with reference and clinically isolated strains of Bordetella pertussis. Simultaneously, we assessed humoral and cellular immunity for evaluating the immunogenicity of a newly developed tri-component diphtheria-tetanus-aP (DTaP) vaccine. Moreover, comparative assessment was made by performing the same evaluations with a commercially available tri-component DTaP vaccine as the positive control. RESULTS: Both groups showed significantly increased levels of antibodies against pertussis toxin, filamentous hemagglutinin and pertactin, and the levels of interferon-γ and interleukin-10 were significantly increased after two doses of vaccination. Furthermore, since cross cell-mediated immune reactivity between the two vaccines was detected, the possibility of interchangeability was indirectly suggested. Although the positive control group showed significantly higher titers in antibody responses for filamentous hemagglutinin and pertactin compared to the experimental group, anti-pertussis toxin antibody titers of the two groups were not significantly different and the protective efficacy against the clinical and reference strains was maintained in both groups for 18 weeks. CONCLUSION: The results showed inferior immunogenicity of the new DTaP vaccine compared to a commercial vaccine despite comparable cellular immunity and protective efficacy. Some efforts are necessary for improving immunogenicity against filamentous hemagglutinin and pertactin before conducting human clinical trials.

Seropositivity of Varicella zoster virus in vaccinated Korean children and MAV vaccine group.

In 2005, a single-dose varicella vaccination was incorporated into the national immunization program in Korea. Although the Oka strain is the most commonly circulating Varicella zoster virus (VZV) genotype in Korea, a domestically manufactured vaccine based on the MAV strain is widely distributed in Korea. High vaccination coverage was achieved, but breakthrough infections were frequently reported. Therefore, in this study, we aimed to analyze the maintenance of immunity after single-dose vaccination and to compare the immunity provided by the MAV and Oka vaccines. Enzyme-linked immunosorbent assays of samples from 715 vaccinated Korean children were used to assess seropositivity rates against VZV. Additionally, fluorescent-antibody-to-membrane-antigen (FAMA) tests were performed in 35 individuals in the MAV vaccine group, and seropositivity rates against the Oka strain were determined. The progressive decrease of the seropositivity rate was demonstrated from ages 1 to 4 as follows: 65% in age 1, 59% in age 2, 53% in age 3, 49% in age 4. It then increased to 62% in age 5 and 70% in age 6. Both the MAV and Oka vaccine groups showed a progressive decrease of the seropositivity rate from the age of 1 to 4 years, but the MAV vaccine group had higher seropositivity rates. In FAMA test, the MAV group demonstrated 71% seropositivity against the Oka strain. This study indicates that immunity wanes after single-dose varicella vaccination and that the MAV vaccine is not inferior to the Oka vaccine in providing immunity against VZV. The MAV vaccine also induced cross-immunity against circulating Oka strain.

Preliminary study on the immunogenicity of a newly developed GCC Tdap vaccine and its protection efficacy against Bordetella pertussis in a murine intranasal challenge model.

PURPOSE: Active reduced dose tetanus-diphtheria-acellular pertussis (Tdap) vaccination for adolescents and adults is necessary because waning immunity after primary diphtheria-tetanus-pertussis vaccination is related to the recent emergence of pertussis. This study was conducted to compare the immunogenicity and protection efficacy against Bordetella pertussis between a new GCC Tdap vaccine and a commercially available Tdap vaccine in a murine model. MATERIALS AND METHODS: BALB/c mice were immunized with two doses of diphtheria-tetanus-acellular pertussis (DTaP) vaccine for priming and a subsequent Tdap booster vaccination. According to the type of booster vaccine, mice were divided into four groups: commercially available Tdap vaccine in group 1 and GCC Tdap vaccines of different combinations of pertussis antigens in groups 2 to 4. Humoral and cell-mediated immune responses and protection efficacy using a murine intranasal challenge model after booster vaccination were compared among the four groups. RESULTS: Every group showed significant increases in antibody titers against pertussis antigens such as pertussis toxin, filamentous hemagglutinin, and pertactin after booster vaccination. Spleen cells showed both Th1 and Th2 cell-mediated immune responses stimulated by pertussis antigens in all groups without any significant difference. In the intranasal B. pertussis infection model, bacteria were eradicated in all groups five days after challenge infection. CONCLUSION: This preliminary study did not show significantly different immunogenicity or protection efficacy of the new GCC Tdap vaccines compared to the commercially available Tdap vaccine, although a more extensive study is necessary to assess the differing efficacies of the new GCC Tdap vaccines.