An Evaluation of the Safety, Immunogenicity, and Protective Efficacy of a Combined Diphtheria-Tetanus-Acellular Pertussis, Haemophilus influenzae Type b, and ACYW135 Meningococcal Conjugate Vaccine in Murine and Rat Models

Abstract

Background: The combined diphtheria-tetanus-acellular pertussis (three-component), Haemophilus influenzae type b (Hib, conjugate), and ACYW135 meningococcal (conjugate) vaccine (DTaP-Hib-MCV4) offers a promising alternative to single-component vaccines, potentially simplifying immunization schedules and improving vaccination coverage. Methods: We evaluated the safety, immunogenicity, and protective efficacy of DTaP-Hib-MCV4 in animal models. Acute and long-term toxicity studies were conducted in Sprague-Dawley (SD) rats with equal numbers of male and female animals. Immunogenicity was assessed in female NIH mice and SD rats using a three-dose regimen at 14-day intervals. Orbital blood was collected 14 days post-immunization to measure IgG titers against pertussis, diphtheria, tetanus, Hib, and meningococcal antigens. The protective efficacy was determined using potency tests for the pertussis, diphtheria, and tetanus components; passive protection studies for Hib; and serum bactericidal antibody (SBA) titers against A/C/Y/W135 meningococcal serogroups. Results: Acute and repeated-dose toxicity studies in SD rats showed no signs of abnormal toxicity or irritation at either high (three doses/rat) or low (one dose/rat) doses levels. The no-observed-adverse-effect level (NOAEL) for DTaP-Hib-MCV4 was established at three doses/rat after 8 weeks of repeated intramuscular administration and a 4-week recovery period. Specific IgG antibodies against all the vaccine components were detected in animal sera at both one and three doses/rat, with no evidence of immunotoxicity. Following two-dose primary immunization in murine models, the combined vaccine elicited robust antigen-specific antibody responses, with geometric mean titers (GMTs) as follows: 1,280,000 for pertussis toxin (PT); 761,093 for filamentous hemagglutinin (FHA); 1,159,326 for pertactin (PRN); 1,659,955 for diphtheria toxoid (DT); 1,522,185 for tetanus toxoid (TT); 99 for Haemophilus influenzae type b (Hib); and 25,600, 33,199, 8300, and 9051 for serogroups A, C, Y, and W135 of Neisseria meningitidis, respectively. In the rat models, three-dose primary immunization also elicited robust antigen-specific antibody responses. Protection studies demonstrated efficacy against pertussis, tetanus toxin, and diphtheria toxin challenges. In the Hib challenge study, none of the 10 animals given anti-DTaP-Hib-MCV4 antiserum developed bacteremia after the live Hib challenge (vs. 5814/0.1 mL in the negative control, p < 0.001). In addition, the SBA titers against meningococcal serogroups exceeded the protective threshold (≥1:8) in 92.2% of the immunized mice and 100% of the immunized rats. Crucially, the combined vaccine induced potent immune responses and protective efficacy, with antibody levels and protection against each component antigen comparable to or greater than those of the individual components: DTaP, Hib, and MCV4. Conclusions: These findings demonstrate that the DTaP-Hib-MCV4 combined vaccine is both safe and immunogenic, supporting its potential as a viable alternative to individual vaccines. This combined vaccine may streamline immunization programs and enhance vaccination coverage.

Keywords: ACYW135 Meningococcal Conjugate Vaccine; Combined Diphtheria-Tetanus-Acellular Pertussis; Haemophilus influenzae Type b; immunogenicity; protective efficacy; safety.

Figure 1

Humoral immune responses of the DTaP-Hib-MCV4 vaccine administered with different doses to NIH mice via the subcutaneous (s.c.) immunization route. The NIH mice were immunized with DTaP-Hib-MCV4 on days 0, 14, and 28, and blood samples were collected on days 14, 28, and 42 before the next vaccine. Serum IgG binding antibodies against DTaP (A), Hib (B), and MCV4 (C) were measured by ELISA. The data represents the geometric mean titer (GMT) ± standard deviation (SD), n = 8 in each group. p values of ≥0.05 indicate no significant difference. When there was no significant difference between the two groups, it was not marked in the picture. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 2

Humoral immune responses to the DTaP-Hib-MCV4 vaccine at different doses in SD rats following s.c. immunization. The SD rats were immunized with DTaP-Hib-MCV4 on days 0, 14, and 28, and blood samples were collected on day 14, 28, and 42 (prior to the next vaccination). Serum IgG binding antibodies against the DTaP group (A), Hib (B), and MCV4 (C) were measured by ELISA. The data presents as the geometric mean titer (GMT) ± standard deviation (SD), n = 8 in each group. p values of ≥0.05 indicate no significant difference. When there was no significant difference between the two groups, it was not marked in the picture. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 3

Humoral immune responses to the DTaP-Hib-MCV4 vaccine compared with those induced by DTaP, Hib, and MCV4 individual vaccines in an NIH mice model via s.c. immunization. The NIH mice were immunized with DTaP-Hib-MCV4, DTaP, Hib, or MCV4 on days 0, 14, and 28, and blood samples were collected on days 14, 28, and 42 (prior to the next vaccination). Serum IgG binding antibodies against DTaP group (A), Hib (B), and MCV4 (C) were measured by ELISA. The data are presented as the geometric mean titer (GMT) ± standard deviation (SD), n = 8 in each group. p values of ≥ 0.05 indicate no significant difference. When there was no significant difference between the two groups, it was not marked in the picture. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 4

The humoral immune response to the DTaP-Hib-MCV4 vaccine compared with DTaP, Hib, and MCV4 individual vaccines in an SD rat model via s.c. immunization. The SD rats were immunized with DTaP-Hib-MCV4, DTaP, Hib, or MCV4 on days 0, 14, and 28, and blood samples were collected on days 14, 28, and 42 (prior to the next vaccination). Serum IgG binding antibodies against the DTaP group (A), Hib (B), and MCV4 (C) were measured by ELISA. The data are represented as the geometric mean titer (GMT) ± standard deviation (SD), n = 8 in each group. p values of ≥0.05 indicate no significant difference. When there was no significant difference between the two groups, it was not marked in the picture. * p < 0.05, ** p < 0.01.

Figure 5

Results of potency test of DTaP vs. DTaP-Hib-MCV4. (IU/SHD = International Unit/Standard Human Dose).

Figure 6

The SBA response to the DTaP-Hib-MCV4 vaccine and the MCV4 vaccine after three doses of immunization. NIH mice (A) and SD rats (B) were immunized with DTaP-Hib-MCV4 or MCV4 on days 0, 14, and 28, and blood samples were collected on day 42. The data are represented as the geometric mean titer (GMT) ± standard deviation (SD), n = 8 in each group. p values of ≥0.05 indicate no significant difference. When there was no significant difference between the two groups, it was not marked in the picture. * p < 0.05, ** p < 0.01, *** p < 0.001.