Immunogenicity of a whole-cell pertussis vaccine with low lipopolysaccharide content in infants

Abstract

The lack of a clear correlation between the levels of antibody to pertussis antigens and protection against disease lends credence to the possibility that cell-mediated immunity provides primary protection against disease. This phase I comparative trial had the aim of comparing the in vitro cellular immune response and anti-pertussis toxin (anti-PT) immunoglobulin G (IgG) titers induced by a cellular pertussis vaccine with low lipopolysaccharide (LPS) content (wP(low) vaccine) with those induced by the conventional whole-cell pertussis (wP) vaccine. A total of 234 infants were vaccinated at 2, 4, and 6 months with the conventional wP vaccine or the wP(low) vaccine. Proliferation of CD3(+) T cells was evaluated by flow cytometry after 6 days of peripheral blood mononuclear cell culture with stimulation with heat-killed Bordetella pertussis or phytohemagglutinin (PHA). CD3(+), CD4(+), CD8(+), and T-cell receptor gammadelta-positive (gammadelta(+)) cells were identified in the gate of blast lymphocytes. Gamma interferon, tumor necrosis factor alpha, interleukin-4 (IL-4), and IL-10 levels in supernatants and serum anti-PT IgG levels were determined using enzyme-linked immunosorbent assay (ELISA). The net percentage of CD3(+) blasts in cultures with B. pertussis in the group vaccinated with wP was higher than that in the group vaccinated with the wP(low) vaccine (medians of 6.2% for the wP vaccine and 3.9% for the wP(low) vaccine; P = 0.029). The frequencies of proliferating CD4(+), CD8(+), and gammadelta(+) cells, cytokine concentrations in supernatants, and the geometric mean titers of anti-PT IgG were similar for the two vaccination groups. There was a significant difference between the T-cell subpopulations for B. pertussis and PHA cultures, with a higher percentage of gammadelta(+) cells in the B. pertussis cultures (P < 0.001). The overall data did suggest that wP vaccination resulted in modestly better specific CD3(+) cell proliferation, and gammadelta(+) cell expansions were similar with the two vaccines.

FIG. 1.

Flow cytometry of unstimulated (control) and B. pertussis- and PHA-stimulated PBMC of a 7-month-old infant vaccinated with a cellular pertussis vaccine with low LPS content. After gating of CD3⁺ cells (A), events were analyzed for size and complexity (forward-scatter and side-scatter gates), from which resting and blast lymphocytes were separated (B). T-lymphocyte subsets (CD4⁺, CD8⁺, and δγ⁺) were then verified in blast lymphocytes.

FIG. 2.

Distribution of CD3⁺ blasts (percentages) determined by flow cytometry of B. pertussis (Bp; ▪)- and PHA (▴)-stimulated PBMC from wP_low vaccine- or wP vaccine-immunized infants. Medians (indicated by bars) of CD3⁺ blasts in B. pertussis-stimulated cultures were 3.9% and 6.2% for wP_low and wP vaccines, respectively. Median percentages of CD3⁺ blasts in PHA-stimulated cultures were 81.4% and 82.5%, respectively. n, number of individuals.

FIG. 3.

IFN-γ (A), TNF-α (B), and IL-10 (C) concentrations determined by ELISA (values are given in picograms per milliliter) by PBMC in cultures without stimuli (control), with B. pertussis (Bp), or with PHA (•) stimulation in infants immunized with wP_low or wP vaccine. Bars indicate medians, and the Mann-Whitney test was used to verify significances. n, number of individuals.