Characterization of humoral and cellular immune responses elicited by meningococcal carriage

Abstract

In order to study the immune response elicited by asymptomatic carriage of Neisseria meningitidis, samples of serum, peripheral blood mononuclear cells (PBMCs), and saliva were collected from a cohort of more than 200 undergraduate students in Nottingham, United Kingdom, who were subject to high rates of acquisition and carriage of meningococci. Serum immunoglobulin G levels were elevated following increases in the rate of carriage, and these responses were specific for the colonizing strains. In order to investigate T-cell responses, PBMCs from 15 individuals were stimulated with a whole-cell lysate of the H44/76 meningococcal strain (B:15:P1.7,16), stained to detect cell surface markers and intracellular cytokines, and examined by flow cytometry. The cells were analyzed for expression of CD69 (to indicate activation), gamma interferon (IFN-gamma) (a representative T-helper 1 subset [Th1]-associated cytokine), and interleukin-5 (IL-5) (a Th2-associated cytokine). Following a brief meningococcal stimulation, the numbers of CD69(+) IFN-gamma(+) CD56/16(+) NK cells were much higher than cytokine-positive CD4(+) events. Both IFN-gamma(+) and IL-5(+) events were detected among the CD69(+) CD4(+) population, leading to the conclusion that an unbiased T-helper subset response was elicited by meningococcal carriage.

FIG. 1.

Relative concentrations of serum antibodies with specificity for the H44/76 meningococcal strain. Serum samples collected from 43 individuals at three time points during the course of the study were tested by indirect ELISA for meningococcus-specific IgG (a), IgA (b), and IgM (c) antibodies. The shaded bars represent the geometric mean concentrations; error bars depict the 95% CIs.

FIG. 2.

Strain specificity of serum antibody responses among a group of 12 confirmed meningococcal carriers. The points and lines represent, for each individual, concentrations of serum IgG with specificity for homologous isolates recovered from pharyngeal swabs compared with those of anti-strain H44/76 IgG. The shaded bars represent the geometric mean concentrations; error bars depict the 95% CIs.

FIG. 3.

Bactericidal activity of serum samples collected from 19 students at week 1 and weeks 20 to 21. Sets of serially diluted serum samples were incubated with 900 CFU of the H44/76 meningococcal strain per reaction well in the presence of human complement. Bactericidal antibody titers were calculated as the serum dilution that yielded a 50% reduction in CFU per well. Shaded bars show the geometric means; error bars represent the 95% CIs.

FIG. 4.

Salivary IgA antibody responses elicited by meningococcal carriage. Saliva samples were collected from 37 students at two time points during the study and stabilized against proteolytic damage. The samples were tested by indirect ELISA for total and H44/76 strain-specific IgA. Shaded bars represent geometric mean ratios of specific to total immunoglobulin; error bars show 95% CIs.

FIG. 5.

Proliferative responses of PBMCs to optimum stimulatory concentrations of a heat-killed whole cell lysate of the H44/76 meningococcal strain. PBMCs were incubated with 1 to 50 μg of the H44/76 strain lysate per ml for 7 days, and [³H]thymidine was included for the final 18 h. SIs were calculated as the ratio of mean counts per minute from antigen-stimulated cells to mean counts per minute from unstimulated cells. The points and lines show the SI values obtained from the PBMCs collected from 15 students at three time points during the study. Shaded bars represent the geometric mean SIs for the group; error bars depict the 95% CIs.

FIG. 6.

Frequencies of activated IFN-γ-producing CD4, CD8, and NK cells following incubation of PBMCs with a meningococcal whole-cell lysate. PBMCs collected at three time points from 15 students were incubated for 10 h in the presence of a heat-killed lysate (20 μg/ml) from the H44/76 strain (░⃞) or medium only (□). The cells were stained for CD4, CD8, and CD16/56 prior to fixation, permeabilization, intracellular staining for CD69 and IFN-γ, and flow cytometry. Error bars depict 95% CIs.

FIG. 7.

Frequencies of activated CD4 cells staining positive for IFN-γ or IL-5 following incubation of PBMCs with a meningococcal whole-cell lysate. The CD4 cytokine responses of PBMCs from 15 students were detected by intracellular staining and flow cytometry following 10 h of incubation with a heat-killed whole-cell lysate of the H44/76 strain. Points represent the individual frequencies of cytokine-positive CD69⁺ CD4⁺ events. Lines depict the median frequency for the group. Stimulating the PBMCs with PMA and ionomycin resulted in >11,000 IFN-γ⁺ and >3,100 IL-5⁺ CD69⁺ CD4⁺ events per 10⁶ cells at each of the time points. Unstimulated PBMCs from weeks 1, 7 to 8, and 20 to 21 contained median frequencies of 95, 92, and 81 IFN-γ⁺ CD4⁺ CD69⁺ events and 43, 0, and 56 IL-5⁺ CD4⁺ CD69⁺ events per 10⁶ cells, respectively.