Background
Bordetella pertussis is the causative agent of pertussis, which is characterized by a paroxysmal cough, and may lead to severe complications and mortality [
1,
2]. Occurrence of pertussis has decreased since the introduction of the diphtheria-tetanus-whole cell pertussis (DTwP) vaccine in the 1950s, which contains pertussis antigens as well as diphtheria and tetanus toxoids [
1]. The diphtheria-tetanus-acellular pertussis (DTaP) vaccine was later developed to reduce the severe adverse effects associated with pertussis antigens included in the DTwP vaccine [
3]. In Korea, DTaP vaccines have replaced DTwP vaccines since 1985 [
4], and in other western countries, DTaP vaccines have been administered since the 1990s [
1,
5]. However, resurgence of pertussis was observed in various western countries after then [
1,
2,
5], and it has also been observed in Korea since the 2000s [
4]. Such resurgence of pertussis is believed to be a consequence of waning immunity against pertussis acquired by DTaP vaccination, and therefore, a booster vaccination with a tetanus-reduced dose diphtheria-acellular pertussis (Tdap) vaccine during adolescence was introduced [
6,
7]. In anticipation of a predicted Tdap vaccine shortage, the Green Cross Corporation (GCC; Yongin, Korea), a pharmaceutical company of Korea, developed a new Tdap vaccine [
8].
The World Health Organization (WHO) recommends conducting non-clinical trials for newly developed vaccines containing acellular pertussis (aP) antigens, if the vaccine contains a novel antigen or is manufactured by a new manufacturer, new process, or new strain [
9]. We previously reported the immunogenicity and protection efficacy of the newly developed GCC Tdap vaccine in a murine model in 2015, and demonstrated that the new vaccine showed comparable efficacy with a commercially available Tdap vaccine [
8]. However, in the previous study, a robust antibody response was observed after two doses of primary DTaP vaccination, which prevented differentiation of the effects of the Tdap booster vaccine [
8].
The present study was performed to evaluate the immunogenicity, protection efficacy, and safety of the new GCC Tdap vaccine in a murine model, using a strategy that addressed the limitations of the previous study. This study will assist in the establishment of future non-clinical trials on Tdap vaccines.
Discussion
In the present study, the immunogenicity and safety of a newly developed Tdap vaccine were determined in a murine model. The new Tdap vaccine showed comparable immunogenicity, protection efficacy, and safety with a commercially available Tdap vaccine.
The WHO recommends a non-clinical study as well as a clinical study before approval and implementation of a new vaccine containing aP antigens [
9]. Accordingly, several non-clinical trials on the immunogenicity, protection efficacy, and safety of DTaP vaccines have been reported [
10,
11,
16,
17]. However, only a few non-clinical trials on Tdap vaccines have been reported as commercially available Tdap vaccines were manufactured using the same aP antigens included in approved DTaP vaccines [
12], and Tdap vaccines were approved based on clinical trial results [
18‐
20]. Although non-clinical trials are necessary for Tdap vaccines manufactured by companies that have not previously produced DTaP vaccines, the methodology of these trials has yet to be established. In particular, it is essential to determine whether Tdap vaccines containing lower doses of diphtheria toxoid and pertussis antigens compared with DTaP vaccines can induce a boosting effect. We have previously reported preliminary results from a non-clinical study of the new GCC Tdap vaccine using a murine model [
8]. In this study, mice received two doses of DTaP vaccine as primary vaccination followed by a single dose of Tdap vaccine as booster vaccination, a method that considered the human vaccination schedule of four doses of DTaP vaccine and one dose of Tdap vaccine [
8]. Although the new Tdap vaccine showed favorable immunogenicity and protection efficacy against
B. pertussis intranasal challenge, antibody titers against pertussis antigens were high even before Tdap booster vaccination, and inoculated
B. pertussis was completely eradicated within 5 days of intranasal challenge [
8]. These results are believed to be a consequence of the prolonged effect of two shots of DTaP vaccination supplementing the boosting effect of Tdap vaccination. As a result, primary DTaP vaccination was administered as a single dose in the present study, and the interval between the primary and booster vaccinations was extended from 6 weeks to 12 weeks.
In the present study, anti-PT IgG and anti-FHA IgG titers significantly increased in the GCC vaccine group (Group 4) and positive control group (Group 5) after Tdap booster vaccination, and no significant difference was observed after booster vaccination between the two groups. Therefore, the immunogenicity of a Tdap vaccine can be determined using one dose of primary DTaP vaccine followed by one dose of Tdap booster vaccine in a murine model, and the new GCC Tdap vaccine showed immunogenicity comparable with a commercially available Tdap vaccine using this strategy. In a previous non-clinical study of a Tdap vaccine in a murine model, one dose of primary DTwP vaccine was administered followed by one dose of Tdap booster vaccine 12 weeks later [
12]. The boosting effect of the Tdap vaccine decreased upon reduction of pertussis antigen dose in the primary DTwP vaccine, and the authors therefore recommended an increased interval between primary and booster vaccinations to rule out residual effects of primary vaccination [
12]. In the present study, the antibody titers against pertussis antigens, DT and TT tended to increase after booster vaccination even in the Groups 2 and 3, in which PBS or Td vaccine was administered as the booster vaccination, although the anti-PT and anti-FHA IgG titers against pertussis antigens in the Groups 2 and 3 were lower than those in the Groups 4 and 5. This result could be caused by the prolonged effects of the primary DTaP vaccination in the Groups 2 and 3. Therefore, the interval between primary and booster vaccinations should be longer than 12 weeks to completely exclude the residual effects of the primary vaccination.
An intranasal challenge test was additionally performed to determine the protection efficacy of the Tdap vaccine against
B. pertussis in the present study, although the WHO recommendations for non-clinical studies only specify antibody tests [
9]. Because
B. pertussis was eradicated within 5 days of intranasal infection in the preliminary study [
8], colony counts were determined 2 and 4 days after infection in the present study. With the use of a single dose of DTaP vaccine, the bacteria were eradicated 7 days after intranasal infection, 2 days later than with two doses. The GCC vaccine group showed significantly lower CFUs than the positive control group 2 days after infection, indicating a potent effect in the early phase. If we consider that the results of the intranasal challenge test in a murine model are representative of clinical efficacy in humans [
11], the new GCC Tdap vaccine may be used effectively in the clinical field. In addition, the new Tdap vaccine showed comparable results with a commercially available Tdap vaccine in MWGT and LP test. Therefore, further clinical trials for the new Tdap vaccine may be performed safely. In mice treated with DTaP vaccine at 25% of the human dose, inoculated
B. pertussis persisted longer than 9 days after intranasal challenge in the negative control group injected with normal saline as a booster vaccination. We thought this dose was sufficiently low to prevent residual effects of the primary DTaP vaccination on the boosting response of the Tdap vaccine, given the 12-week interval between primary and booster vaccinations; however, an interval longer than 12 weeks could be appropriate to exclude the residual effects, considering the results of the immunogenicity assessment.
This study has several limitations. Neither the GCC nor the commercial Tdap vaccine had a significant effect on anti-PRN IgG titers. However, significant humoral immune responses to PT and FHA and a significant protection efficacy in the intranasal challenge model were observed in both groups. Although cellular immune responses also play an important role in the protective immunity against
B. pertussis [
17,
21‐
23], they were not measured in the present study. In previous studies, Th1 and Th17 responses were detected after DTwP vaccination and natural
B. pertussis infection, and Th2 responses were detected after DTaP vaccination [
17,
21,
22,
24]. In contrast, some investigators reported Th1-dominant responses or Th1 and Th2 mixed responses even after DTaP vaccination [
16,
25]. Moreover, cellular immune responses after booster DTaP or Tdap vaccination differed according to the type of primary vaccination in some studies [
12,
26]. Therefore, future studies should determine cellular immune responses elicited by Tdap booster vaccination. Based on the results of cellular immune responses of the new Tdap vaccine with a combination of the results of the present study, clinical trials should be planned. For in vivo assessment of residual toxicity of aP antigens, a histamine sensitization test is recommended [
9]. The present study instead used MWGT and LP test. The histamine sensitization test is supposed to be performed using a GCC DTaP vaccine containing an increased aP antigen dose than the GCC Tdap vaccine.