Contributions of capsule, lipoproteins and duration of colonisation towards the protective immunity of prior Streptococcus pneumoniae nasopharyngeal colonisation

Abstract

Live attenuated vaccines have been proposed as a strategy to induce protective immunity against infectious diseases. Recent data have demonstrated that nasopharyngeal colonisation with Streptococcus pneumoniae induces protective immunity against subsequent invasive infection, suggesting nasal vaccination with live attenuated bacteria could be a preventative strategy. However the bacterial factors affecting the strength of this adaptive immune response remain unclear. In a direct comparison with the parent wild-type strain, we found that colonisation with bacteria lacking either capsule or surface lipoproteins led to significantly diminished protection. Immunity after colonisation was not dependent on serum IgG to capsular antigens. Colonisation density and duration was reduced for all the non-protective strains, suggesting that protective immunity maybe related to the extent of nasopharyngeal bacterial exposure. To investigate this hypothesis, we utilised an auxotrophic bacterial Δpab strain where duration of colonisation could be controlled by supply and removal of para-amino-benzoic acid (PABA) to mouse drinking water. Supporting colonisation with the Δpab strain for 5 days with PABA led to a faster serum antibody response compared to colonisation for less than 48 h. This enhanced immunogenicity was associated with a trend towards protection. The data presented here aid our understanding of why only certain live attenuated strains are able to function as effective vaccines, and may be valuable in informing the constituents of future live attenuated vaccines.

Fig. 1

Protective effect of prior colonisation on subsequent S. pneumoniae pneumonia. Kaplan–Meier survival curves of mice challenged with 10⁷ cfu of D39 i.n., 28 days after colonisation with either one or two (×2) doses of 10⁷ cfu of D39, D39-DΔ, D39Δlgt, D39Δpab or PBS alone (n = 18–20). *P < 0.05, ***P < 0.001.

Fig. 2

Serum antibody response to colonisation. Serum IgG (A), IgA (B) and IgM (C) responses to colonisation with either one or two (×2) doses of D39, D39-DΔ, D39Δlgt or PBS alone, measured using whole cell ELISA. Dots indicate responses of individual mice, bars represent mean for the group. *P < 0.05, ***P < 0.001.

Fig. 3

Correlation of immunogenicity with protection following nasopharyngeal colonisation. Coloured dots and bars represent immunogenicity as anti-D39WT IgG geomean titre (GMT) of the group (±95% CI) versus percentage survival (±95% CI) of the group against day 28 D39WT pneumonia challenge. Dotted line shows correlation between immunogenicity and protection by linear regression.

Fig. 4

Serum anti-capsular IgG response to colonisation. (A) Serum anti-type 2 capsular polysaccharide IgG response to colonisation with either one or two (×2) doses of D39, D39-DΔ, D39Δlgt, D39Δpab or PBS alone, measured by ELISA. Dots indicate responses of individual mice, bars represent mean for the group. (B) Competitive inhibition of binding of serum IgG to total D39 antigens in whole cell ELISA using increasing concentrations of soluble type 2 capsular polysaccharide. Bars represent relative mean ± SD of binding of sera from individual mice (n = 4).

Fig. 5

Serum anti-protein IgG responses to colonisation. IgG binding of sera of three individual WT D39 colonised mice to S. pneumoniae proteins measured by Luminex bead assay. Dotted line represents limit of detection.

Fig. 6

Timecourse of colonisation with S. pneumoniae D39 strains. Bacterial cfu recovered from nasal washes of CD1 mice on days 1, 2, 5, 10 and 17 following intranasal inoculation in 10 μl of PBS containing 10⁷ cfu D39 S. pneumoniae (closed squares) or D39-DΔ (open squares), D39Δpab (closed circles) or D39Δlgt (open circles). Median and interquartile range of groups of 4–9 mice at each timepoint are shown, along with the precise number in the group. The dotted line is the limit of detection.

Fig. 7

Effect of para-aminobenzoic acid (PABA) supplementation on immunogenicity and protection. (A) Bacterial cfu recovered from nasal washes on days 1, 2, 5 and 7 following intranasal inoculation with 10⁷ cfu D39Δpab in the presence of PABA supplementation. PABA was withdrawn on day 5. Dots represent results for individual mice, bards represent group median. The dotted line is the limit of detection. (B) Serum anti-D39 IgG level measured by whole cell ELISA at 0, 14 and 28 days following colonisation with (black circles) or without (white circles) 5 days of PABA supplementation. Bars indicate group mean. *P < 0.05. (C) Survival following challenge with 10⁷ cfu D39 i.n. 28 days after colonisation with D39Δpab with or without 5 days of PABA supplementation, or with PBS alone (n = 15).