Safety assessments of recombinant DTaP vaccines developed in South Korea.

Purpose: Pertussis bacteria have many pathogenic and virulent antigens and severe adverse reactions have occurred when using inactivated whole-cell pertussis vaccines. Therefore, inactivated acellular pertussis (aP) vaccines and genetically detoxified recombinant pertussis (rP) vaccines are being developed. The aim of this study was to assess the safety profile of a novel rP vaccine under development in comparison to commercial diphtheria-tetanus-acellular pertussis (DTaP) vaccines. Materials and Methods: The two positive control DTaP vaccines (two- and tri-components aP vaccines) and two experimental recombinant DTaP (rDTaP) vaccine (two- and tri-components aP vaccines adsorbed to either aluminum hydroxide or purified oat beta-glucan) were used. Temperature histamine sensitization test (HIST), indirect Chinese hamster ovary (CHO) cell cluster assay, mouse-weight-gain (MWG) test, leukocytosis promoting (LP) test, and intramuscular inflammatory cytokine assay of the injection site performed for safety assessments. Results: HIST results showed absence of residual pertussis toxin (PTx) in both control and experimental DTaP vaccine groups, whereas in groups immunized with tri-components vaccines, the experimental tri-components rDTaP absorbed to alum showed an ultra-small amount of 0.0066 IU/mL. CHO cell clustering was observed from 4 IU/mL in all groups. LP tests showed that neutrophils and lymphocytes were in the normal range in all groups immunized with the two components vaccine. However, in the tri-components control DTaP vaccine group, as well as two- and tri-components rDTaP with beta-glucan group, a higher monocyte count was observed 3 days after vaccination, although less than 2 times the normal range. In the MWG test, both groups showed changes less than 20% in body temperature and body weight before the after the final immunizations. Inflammatory cytokines within the muscle at the injection site on day 3 after intramuscular injection revealed no significant response in all groups. Conclusion: There were no findings associated with residual PTx, and no significant differences in both local and systemic adverse reactions in the novel rDTaP vaccine compared to existing available DTaP vaccines. The results suggest that the novel rDTaP vaccine is safe.

Comparative Evaluation of Recombinant and Acellular Pertussis Vaccines in a Murine Model.

Since the 2000s, sporadic outbreaks of whooping cough have been reported in advanced countries, where the acellular pertussis vaccination rate is relatively high, and in developing countries. Small-scale whooping cough has also continued in many countries, due in part to the waning of immune protection after childhood vaccination, necessitating the development of an improved pertussis vaccine and vaccination program. Currently, two different production platforms are being actively pursued in Korea; one is based on the aP (acellular pertussis) vaccine purified from B. pertussis containing pertussis toxoid (PT), filamentous hemagglutin (FHA) and pertactin (PRN), and the other is based on the recombinant aP (raP), containing genetically detoxified pertussis toxin ADP-ribosyltransferase subunit 1 (PtxS1), FHA, and PRN domain, expressed and purified from recombinant E. coli. aP components were further combined with diphtheria and tetanus vaccine components as a prototype DTaP vaccine by GC Pharma (GC DTaP vaccine). We evaluated and compared the immunogenicity and the protective efficacy of aP and raP vaccines in an experimental murine challenge model: humoral immunity in serum, IgA secretion in nasal lavage, bacterial clearance after challenge, PTx (pertussis toxin) CHO cell neutralization titer, cytokine secretion in spleen single cell, and tissue resident memory CD4+ T cell (CD4+ TRM cell) in lung tissues. In humoral immunogenicity, GC DTaP vaccines showed high titers for PT and PRN and showed similar patterns in nasal lavage and IL-5 cytokine secretions. The GC DTaP vaccine and the control vaccine showed equivalent results in bacterial clearance after challenge, PTx CHO cell neutralization assay, and CD4+ TRM cell. In contrast, the recombinant raP vaccine exhibited strong antibody responses for FHA and PRN, albeit with low antibody level of PT and low titer in PTx CHO neutralization assay, as compared to control and GC DTaP vaccines. The raP vaccine provided a sterile lung bacterial clearance comparable to a commercial control vaccine after the experimental challenge in murine model. Moreover, raP exhibited a strong cytokine response and CD4+ TRM cell in lung tissue, comparable or superior to the experimental and commercial DTaP vaccinated groups. Contingent on improving the biophysical stability and humoral response to PT, the raP vaccine warrants further development as an effective alternative to aP vaccines for the control of a pertussis outbreak.

Seroprevalence of varicella-zoster virus as measured by fluorescent antibody to membrane antigen assay and glycoprotein enzyme-linked immunosorbent assay more than 10 years after initiation of a universal vaccination program: An observational study.

Universal varicella vaccination (UVV), as a single dose to children aged 12 to 15 months, was introduced in Korea in 2005. A seroprevalence study is required to upgrade this UVV strategy. The fluorescent antibody to membrane antigen (FAMA) assay is the gold standard for varicella-zoster virus (VZV) immunity testing. However, no standard operating procedure (SOP) has been developed for the FAMA assay, in which either glutaraldehyde or acetone may be used for VZV-infected cell fixation. In this observational study, we aimed to investigate the age-specific seroprevalence in Korean children and adults. Additionally, with glycoprotein enzyme-linked immunosorbent assay (gpELISA) as the reference, we evaluated the performance of the FAMA assay using acetone-fixed cells. Four hundred sera were analyzed using the FAMA assay (acetone-fixed cells) and gpELISA, comprising 50 subjects from each age category. In the FAMA assay, the seropositivity rate decreased from 82.0% in the 1 to 4-year-old group to 58.0% in the 5 to 9-year-old group (95% confidence interval [CI]: 69.2-90.2 and 44.2-70.6, respectively; P = .009), while that in the gpELISA decreased from 80.0% to 52.0% (95% CI: 67.0-88.8 and 38.5-65.2, respectively; P = .003). In both methods, the seropositivity rates ranged from 95% to 100% in the population aged ≥ 20 years. We observed a significant correlation between the 2 methods, with a correlation coefficient of 0.795 (P < .001). In receiver operating characteristic analysis using the gpELISA results as a reference, the area under the curve for the FAMA assay was very high at 0.995 (95% CI: 0.990-1.000; P < .001). Compared to the gpELISA, the sensitivity, specificity, and kappa value of the FAMA assay were 99.4%, 79.3%, and 0.84 (nearly perfect), respectively. The seropositivity rate of the 5 to 9-year-old group indicated waning immunity over time and supported implementation of a second dose in the UVV program. The results of the FAMA assay were comparable to those of the gpELISA. Although further study is needed to standardize procedures, our results suggest that the FAMA assay using acetone-fixed cells can be used widely and can be included in a universal FAMA assay SOP.

A booster administration of the OKA/SK strain causes fatal disseminated varicella in an immunocompetent child.

Live varicella vaccines are known to provide robust immunity against varicella zoster virus (VZV) infections. However, problems with viral attenuation have led to pathogenic VZV vaccine strains causing varicella-like rash and herpes zoster in immunocompetent children after immunization. We report the first fatal case of VZV infection caused by OKA/SK strain contained in the vaccine administrated as a booster shot in an immunocompetent child, which has been independently developed from any currently available varicella vaccines that are OKA strain or MAV/06 strain based. The patient died due to sudden pulmonary alveolar hemorrhage as a secondary complication of VZV pneumonitis. Sequencing of the four SNPs unique to the OKA/SK strain (SNP loci 14 035T; 32 626C; 58 777G; 70 319G) enabled discrimination of the strain responsible for the disseminated infection. OKA/SK strain does not have any SNPs in ORF62 postulated to be responsible for the attenuation of varicella vaccines which have been safely and effectively used world-wide or locally, and exclusively enriches a virulent factor in ORF31 identified in parental OKA strain, thus possibly resulting in disseminated VZV infection leading to mortality. Therefore, actions need to be taken to prevent vaccine related morbidity and mortality in children.

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.

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.

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.

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.