Quantitative analysis of pertussis, tetanus, and diphtheria antibodies in sera and breast milk from Tdap vaccinated women using a qualified multiplex assay

Abstract

Pertussis (whooping cough) is a reemergent, highly contagious respiratory infection of public health concern. Infants prior to initiation of their primary vaccination series are the most vulnerable to severe infection, and even death. Vaccination during pregnancy is an efficacious means of reducing infection in infants. This approach relies on boosting maternal immunity and passive transfer of antibodies to the infant via placenta and breast milk. Similarly, maternal vaccination post-partum can enhance maternal-infant immunity. To support the analysis of pertussis immunity in the context of maternal-infant immunization, we developed a high throughput multiplex assay for simultaneous quantification of serum IgG antibodies against pertussis vaccine antigens: pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN), and fimbriae (FIM2/3), and against tetanus (TT) and diphtheria toxoids (DT), using the Meso Scale Discovery (MSD) platform. The assay was qualified, and specificity, sensitivity, accuracy, precision, linearity, and robustness were demonstrated. The assay was subsequently adapted for quantification of IgG and IgA in breast milk. Applied to a serological survey of pregnant women living in the United States and sub-Saharan Africa, this method revealed differences in magnitude and breadth of antibody profile, consistent with history of vaccination. A longitudinal analysis of Tdap responses in women vaccinated post-partum demonstrated a rapid increase in serum IgG that remained elevated for up to 24 months. Likewise, high levels of vaccine-specific IgA and IgG antibodies were present in breast milk, although they exhibited faster decay. This multiplex MSD assay is a reliable and practical tool for quantification of pertussis, tetanus, and diphtheria antibodies in serum and breast milk in serosurveys or vaccine studies.

Importance: Pertussis (whooping cough) has reemerged in recent years. Vaccination during pregnancy is an effective approach to prevent illness during the first months of life. We developed a multiplex assay for quantification of pertussis, tetanus, and diphtheria serum antibodies using the Meso Scale Discovery (MSD) platform; the method was qualified, and specificity, precision, accuracy, linearity, and limits of quantification were defined. It was also adapted for quantification of antibodies in breast milk. We successfully determined serostatus in women from different regions and with different vaccination histories, as well as responses to Tdap in blood and breast milk post-partum. This is the first description of a multiplex assay for the quantification of pertussis, tetanus, and diphtheria antibodies in breast milk.

Keywords: Tdap; infant immunity; infant vaccines; maternal vaccines; multiplex; pertussis.

Fig 1

Dose–response curves for pertussis, diphtheria, and tetanus antibodies in the International and in-house standards measured by the multiplex assay. (A) WHO International Standard Pertussis (NIBSC 06/140), Tetanus Immunoglobulin (NIBSC TE-3), Diphtheria Antitoxin (NIBSC 10/262), and B. pertussis human serum (NIBSC 89/530); (B) in-house serum IgG standard; (C) in-house breast milk (BM) IgG standard; and (D) in-house BM IgA standard. Data depict mean electrochemiluminescence (ECL) units from duplicate wells versus antibody concentrations for PT (black), PRN (purple), FHA (light blue), FIM 2/3 (light purple), DT (pink), and TT (teal). Representative curves are shown. DT, diphtheria toxoid; FHA, filamentous hemagglutinin; FIM, fimbriae; PT, pertussis toxin; PRN, pertactin; TT, tetanus toxin. Curve parameters, given or assigned unitage, acceptance criteria, and detection limits are shown in Table 1

Fig 2

Serum IgG levels against PT, PRN, FHA, FIM 2/3, DT, and TT in pregnant women with different vaccination histories. Data represent antibody titers in women living in the United States and immunized with Tdap during pregnancy (n = 15), and women living in Malawi who received only tetanus vaccination (not Tdap or Td) during pregnancy (n = 26). Dotted lines represent protective thresholds. *P < 0.05, **P < 0.01, ***P < 0.001 by Kruskal-Wallis H test followed by the Dunn’s pairwise comparison with Sidák adjustment for multiple comparisons. DT, diphtheria toxoid; FHA, filamentous hemagglutinin; FIM, fimbriae; PRN, pertactin; PT, pertussis toxin; TT, tetanus toxin.

Fig 3

Serum IgG levels against PT, PRN, FHA, FIM 2/3, DT, and TT in post-partum women immunized with Adacel Tdap. Women were vaccinated 1–4 days after delivery. Data represent antibody titers before (0) and 0.5–24 months after post-partum Tdap vaccination. Fifty-three women provided samples at day 0; 34 remained in the study and provided blood at 24 months. Dotted lines represent protective thresholds, when available. ****P < 0.0001 by Wilcoxon signed-rank test. DT, diphtheria toxoid; FHA, filamentous hemagglutinin; FIM, fimbriae; PRN, pertactin; PT, pertussis toxin; TT, tetanus toxin.

Fig 4

Breast milk IgG and IgA antibodies against PT, PRN, FHA, DT, and TT in post-partum women immunized Adacel Tdap. Data represent antibody titers 0.5–6 months after post-partum Tdap vaccination. Breast milk samples were available for testing from 18 women at 2 weeks, 31 women at 6 weeks, and 19 women at 6 months. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Wilcoxon signed-rank test. DT, diphtheria toxoid; FHA, filamentous hemagglutinin; FIM, fimbriae; ns, not significant; PRN, pertactin; PT, pertussis toxin; TT, tetanus toxin.

Fig 5

Association between serum and breast milk (BM) antibody levels in post-partum women immunized with Adacel Tdap. Correlation analysis of peak serum and breast milk IgG antibody titers (0.5 months post-partum) against Tdap antigens: (A) PT, (B) PRN, (C) FHA, (D) DT, and (E) TT. In-picture plots magnify data points and correlation at the lower end of the scale. DT, diphtheria toxoid; FHA, filamentous hemagglutinin; PRN, pertactin; PT, pertussis toxin; TT, tetanus toxin. P value and r = Spearman correlation are indicated.