Cell-mediated immune responses in four-year-old children after primary immunization with acellular pertussis vaccines

Abstract

Cell-mediated immune (CMI) responses to Bordetella pertussis antigens (pertussis toxin [PT], pertactin [PRN], and filamentous hemagglutinin [FHA]) were assessed in 48-month-old recipients of acellular pertussis [aP] vaccines (either from Chiron-Biocine [aP-CB] or from SmithKline Beecham [aP-SB]) and compared to CMI responses to the same antigens at 7 months of age, i.e., 1 month after completion of the primary immunization cycle. None of the children enrolled in this study received any booster of pertussis vaccines or was affected by pertussis during the whole follow-up period. Overall, around 75% of 4-year-old children showed a CMI-positive response to at least one B. pertussis antigen, independently of the type of aP vaccine received, and the proportion of CMI responders were at least equal at 48 and 7 months of age. However, longitudinal examination of individual responses showed that from 20 (against PT) to 37% (against FHA) of CMI responders after primary immunization became negative at 48 months of age. This loss was more than compensated for by conversion to positive CMI responses, ranging from 36% against FHA to 69% against PRN, in other children who were CMI negative at 7 months of age. In 60 to 80% of these CMI converters, a lack of decline or even marked elevation of antibody (Ab) titers against B. pertussis antigens also occurred between 20 and 48 months of age. In particular, the frequency of seropositivity to PRN and FHA (but not to PT) was roughly three times higher in CMI converters than in nonconverters. The acquisition of CMI response to B. pertussis antigens in 48-month-old children was not associated with a greater frequency of coughing episodes lasting >/=7 days and was characterized by a prevalent type 1 cytokine profile, with high gamma interferon and low or no production of interleukin-5, reminiscent of cytokine patterns following immunization with whole-cell pertussis vaccine or natural infection. Our data imply that vaccination-induced systemic CMI may wane by 4 years of age but may be acquired or naturally boosted by symptomless or minor clinical infection by B. pertussis. This might explain, at least in part, the persistence of protection against typical pertussis in aP vaccine recipients despite a substantial waning of both Ab and CMI responses induced by the primary immunization.

FIG. 1

Cross-sectional analysis of percent positivity (CMI⁺) (top panels) and magnitude (mean counts per minute ± SE) of proliferative responses (bottom panels) to B. pertussis antigens in aP-CB and aP-SB vaccine recipients at the indicated ages. Proliferation assays were performed by using frozen and fresh PBMC in 7- and 48-month-old children, respectively. In both cases, PBMC (2 × 10⁵/well in 0.2 ml) were cultured in the presence of PT (10 μg/ml), FHA (20 μg/ml), and PRN (20 μg/ml). DNA synthesis was measured after 7 days by counting incorporation. A CMI⁺ response was that occurring in the antigen-stimulated PBMC culture with a [³H]thymidine incorporation value of ≥3,000 cpm with respect to that of the antigen-unstimulated control culture. No statistically significant differences were noticed either between the responses to each antigen at the two ages or between the aP-CB and aP-SB recipients.

FIG. 2

Longitudinal CMI analysis. PBMC (2 × 10⁵/well in 0.2 ml) from 21 aP-CB (A) and 20 aP-SB (B) recipients were assayed for proliferation at 7 and 48 months of age, as indicated. The magnitudes of cell proliferation induced by PT, PRN, and FHA antigens are shown. DNA synthesis was measured after 7 days by counting [³H]thymidine incorporation. Data are expressed as counts per minute of the differences between the antigen-stimulated PBMC culture and the unstimulated culture. On the right of the middle panels, the codes of the subjects tested are given.

FIG. 3

Serum antibody titers at 7, 20, and 48 months of age against B. pertussis antigens indicated in each panel in children who were CMI⁻ at 7 months of age and acquired (CMI⁺; left panels) or did not acquire (CMI⁻; right panels) CMI at 48 months of age. Seropositivity was defined as a value of ELISA units four times higher than the MLD, which was set at 2 U/ml for IgG to PT and FHA and 3 U/ml for IgG to PRN. For the determination of Ab titers and other technical details, see the text. The numeric codes individuate all children tested.

FIG. 4

IFN-γ, IL-5, and TNF-α production in B. pertussis antigen-stimulated PBMC of 48-month-old vaccine recipients. There was an average of 21 (13 CMI⁺ [top panels] and 8 CMI⁻ [bottom panels]) aP-CB and 13 (8 CMI⁺ and 5 CMI⁻) aP-SB recipients. PBMC from CMI⁺ and CMI⁻ children were cultured in the presence of PT and PRN; after 48 h, the supernatants were collected and cytokine production was assayed by ELISA. The data are expressed as means ± SE. All differences in IFN-γ production between data in the top panels and bottom panels for both PRN and PT and both vaccines were highly significant (P < 0.01; Student’s t test). All comparisons of IL-5 and TNF-α production gave nonsignificant differences.