Impaired innate and adaptive immunity to Streptococcus pneumoniae and its effect on colonization in an infant mouse model

Abstract

Streptococcus pneumoniae colonization and invasive disease peak around the third and first birthdays, respectively, and decline thereafter. While these declines are attributable in part to immunity acquired via natural exposure, maturation of innate immune responses may also be involved. A mucosally administered candidate whole-cell pneumococcal vaccine (WCV) containing killed pneumococcal antigen (WCA) plus a cholera toxin adjuvant protects against intranasal carriage of pneumococci by a mechanism that is antibody independent and CD4(+) TH17 cell dependent. Because infants and children are a key target population for this vaccine, we sought to evaluate the immune responses of neonatal and infant mice to S. pneumoniae and to assess whether the WCV would be effective in these mice. Like human infants, infant mice showed impaired clearance of nasopharyngeal colonization with S. pneumoniae. Macrophages from neonatal and infant mice stimulated with killed pneumococci in vitro showed significantly reduced cytokine production, including that of KC, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, macrophage chemoattractant protein 1, interleukin-6 (IL-6), IL-1alpha, tumor necrosis factor alpha, and gamma interferon, whereas IL-10 expression was significantly increased compared to that in macrophages from adult mice. IL-17A production from adult immune CD4(+) T cells was significantly delayed when neonatal macrophages instead of adult macrophages were used as antigen-presenting cells. Moreover, whole blood from mice immunized as neonates with WCV produced significantly less IL-17A after stimulation with WCA than did blood from mice immunized as adults. Nonetheless, a single immunization of neonatal mice with WCV significantly reduced colonization density. Overall, our data suggest an impairment of both innate and acquired cellular immune responses in neonatal and infant mice. However, WCV confers a significant reduction in colonization following pneumococcal challenge, suggesting that it may still be effective in the setting of immature immune responses.

FIG. 1.

Effect of age on duration and density of pneumococcal colonization with a serotype 6B strain in mice. Median colonization densities are depicted 7, 14, and 21 days after intranasal inoculation of infant (14-day-old) and adult (5- to 6-week-old) mice. Each symbol represents the density of colonization of an individual animal. Each line represents the median colonization density in each group. The density of colonization was significantly higher in infant mice examined 7 and 14 days after exposure, and there was a trend toward a longer duration of colonization in infants (P = 0.081).

FIG. 2.

Effect of age on cytokine responses of spleen macrophages stimulated for 24 h with unencapsulated pneumococci. Data here represent the mean plus standard error of the mean (SEM) cytokine responses of macrophages obtained from neonatal, infant, and adult mice. For most samples, triplicate samples were available and were examined by ELISA or Luminex assay. For all cytokines except IL-10, the cytokine production in neonatal macrophages was significantly lower than that in adults. In contrast, IL-10 was consistently higher in neonatal macrophages stimulated with WCA than in adults. *, P < 0.05; **, P < 0.01; #, single value.

FIG. 3.

Time courses of cytokine production of neonatal, infant, and adult macrophages after stimulation with pneumococcal WCA. The mean and SEM cytokine concentrations from triplicate wells (duplicate wells at 14 h) are depicted. Adult macrophages (open circles) produced significantly more IFN-γ, IL-1α, and GM-CSF than did macrophages from neonatal (closed squares) or infant (closed triangles) mice at several time points during the 48-hour stimulation. In contrast, the IL-10 production of macrophages from both neonatal and infant mice upon stimulation with WCA was significantly higher than that of macrophages from adult mice. *, significant difference for neonatal compared to adult macrophages; **, significant difference for both neonatal and infant macrophages compared to adult macrophages.

FIG. 4.

Cytokine production of infant and adult macrophages stimulated for 24 h with the TLR2 agonists MALP-2 and heat-killed L. monocytogenes (HKLM) and the TLR4 agonists PdT and LPS. The mean cytokine concentrations for triplicate wells are depicted, with SEM. Triplicate samples were examined by ELISA or Luminex assay. For all cytokines except IL-10, the cytokine production in neonatal macrophages was significantly lower than that in adult macrophages. In contrast, IL-10 was consistently higher in neonatal macrophages than in adult macrophages for most stimuli. *, P < 0.05; **, P < 0.01.

FIG. 5.

IL-17 production by CD4⁺ T cells from vaccinated adult mice stimulated with WCA at 48 and 72 h in the presence of neonatal or adult macrophages. The mean and SEM cytokine concentrations of triplicate wells are depicted. At 48 h, CD4⁺ T cells showed significantly less expression of IL-17A when neonatal instead of adult macrophages were used as APCs. At 72 h, this difference in IL-17A expression was not significant.

FIG. 6.

Effect of IL-10 on IL-17 production of splenocytes from immunized adult mice. (A) Splenocytes from immunized adult mice were stimulated with WCA in the presence of recombinant IL-10 at the indicated concentrations. IL-17A production measured by ELISA was significantly decreased at 48 h when the highest concentration of IL-10 was added. The mean cytokine concentrations for three replicates are depicted. (B) The reduction in IL-17A production in the presence of 1,000 pg/ml of IL-10 was no longer significant after 72 h of incubation.

FIG. 7.

Effect of age at time of intranasal immunization with WCV on generation of serum antipneumococcal IgG antibodies. The mean IgG antipneumococcal antibody titer in sera from mice immunized as neonates was significantly lower than that for mice immunized as adults. The mice represented by the open symbols were fully protected against colonization by WCV, and the closed symbols represent immunized mice that were still colonized at 7 days postchallenge. The median IgG concentration in five to seven mice per group is depicted in arbitrary units/ml.

FIG. 8.

Effect of age on the efficacy of intranasal immunization with WCV on subsequent nasopharyngeal colonization with serotype 6B pneumococci in mice. A single dose of intranasal WCV was administered to neonates or adults. All mice were challenged with a type 6B pneumococcal strain 6 weeks after immunization. The density of nasopharyngeal colonization was evaluated 1 week later. Each symbol represents the density of colonization of an individual animal. Each line represents the median colonization density in each group. Immunization with WCV resulted in a significant reduction in density of pneumococcal colonization compared to that in control CT-immunized mice for mice immunized as both neonates and adults.