Experimental Human Challenge Defines Distinct Pneumococcal Kinetic Profiles and Mucosal Responses between Colonized and Non-Colonized Adults

Kinetics of pneumococcal detection in nasal fluid following exposure. (A) Frequency of S. pneumoniae (Spn) DNA detection in nasal fluid after challenge, stratified by final colonization status (determined by conventional bacterial culture methods). NLF samples were collected before (T = 0) pneumococcal exposure and at 4, 8, 24, and 48 h post exposure. The number of volunteers with S. pneumoniae 6B presence in each time point is expressed as a percentage (%) of the total number of volunteers analyzed for culture-positives (red) and culture-negatives (black). (B) Individual-level DNA detection results, grouped by culture-positives (red, n = 20) and culture-negatives (n = 39). The latter group is subdivided into saliva clearers (blue, n = 15) and nasal clearers (green, n = 24). Samples not taken are highlighted in gray. (C) Density levels of pneumococcal 6A/B PCR in NLF, expressed as DNA copies per nasosorption device. If no S. pneumoniae was detected, the density was set as 0 CFU/ml. Data was log transformed after adding 1 to all values to allow transforming 0 values. Individual volunteers and the mean of log-transformed values are shown. (D) Levels of agglutination capacity in nasal wash at baseline in initial cohort. Nine culture-positive, 12 saliva clearer, and 17 nasal clearer volunteers were included in analysis. Each dot represents one volunteer. Mean ± standard error of the mean (SEM) is shown for each of the three groups.

Association of baseline mucosal immune factors with early pneumococcal colonization profiles. (A) Neutrophil number in nasal scrapes prior to pneumococcal exposure. Abundance of CD66b-high cells (activated granulocytes) at baseline were measured by flow cytometry. Eight culture-positives, 9 saliva clearers, and 14 nasal clearers were assessed (*, P = 0.018, unpaired t test). There is no statistically significant difference of the CD66b^Hi neutrophil counts between nasal and saliva clearers (P = 0.107, unpaired t test). Data were log transformed, and individual volunteers and mean ± SEM are represented. (B) MPO levels in nasal wash prior to pneumococcal exposure—both cohorts. MPO levels at baseline were measured by ELISA. Twenty-one culture-positives, 16 saliva clearers, and 24 nasal clearers were assessed. MPO levels in nasal wash were significantly increased in nasal clearers compared to those in culture-positives (*, P = 0.034, unpaired t test). Data were log transformed, and individual volunteers and mean ± SEM are represented. (C) Correlation between MPO levels in nasal wash and number of activated granulocytes (CD66b^Hi) in nasal scrapes prior to S. pneumoniae challenge for paired samples. Eight culture-positives, 9 saliva clearers, and 14 nasal clearers were assessed (Pearson test, r = 0.480; **, P = 0.006). Data were log transformed. Culture-positives, red circles; saliva clearers, blue rectangular; and nasal clearers, green squares. (D) MUC5AC in nasal wash at baseline. Each dot represents a volunteer, and mean ± SEM are shown. No statistical significance was detected within the groups (unpaired t test, culture-positives versus saliva clearers, P = 0.568; culture-positives versus nasal clearers, P = 0.112; saliva clearers versus nasal clearers, P = 0.250). (E) Levels of S. pneumoniae 6B polysaccharide-specific IgG (PS6B) antibodies in nasal wash at baseline. Each dot represents a volunteer, and mean ± SEM are shown. No statistical significance was detected within the groups (Mann-Whitney test, culture-positives versus saliva clearers, P = 0.722; culture-positives versus nasal clearers, P = 0.118; saliva clearers versus nasal clearers, P = 0.438). (F) Agglutination capacity (%) versus mucin (MUC5AC) in nasal wash at baseline. Culture-positives, red circles (n = 9); saliva clearers, blue rectangular (n = 7); and nasal clearers, green squares (n = 12). Data were log transformed, and Spearman rho and P values are depicted in addition to linear regression (black line). (G) Agglutination capacity (%) versus S. pneumoniae 6B polysaccharide-specific IgG (PS6B) antibody levels in nasal wash at baseline. Culture-positives, red circles (n = 9); saliva clearers, blue rectangular (n = 12); and nasal clearers, green squares (n = 17). Data were log transformed, and Spearman rho and P values are indicated.

Nasal immune factors during the first 48 h post exposure. (A) MPO levels in nasal lining fluid during the first 48 h measured by ELISA. Volunteer numbers assessed are indicated above the x axis per time point after exposure. Data were log transformed, and individuals and mean ± SEM are depicted. In culture-positives and saliva clearers, levels of MPO rose after exposure, peaking at 24 h after pneumococcal exposure (*, P = 0.016 and **, P = 0.006, respectively; paired t test to baseline). MPO baseline levels are statistically significantly different between nasal clearers and saliva clearers (*, P = 0.046, unpaired t test). (B) Cytokine profile heatmap in nasal lining fluid following S. pneumoniae challenge within the three groups. Concentrations of 30 cytokines was measured by Luminex at 4, 8, 24, and 48 h after pneumococcal exposure and normalized to baseline levels for each subject. The mean of log₂-tranformed fold changes are shown per time point for each of the three groups. *, P < 0.05 based on paired t test comparing to baseline, followed by multiple testing correction (Benjamini-Hochberg). (C) Cytokine induction score in nasal lining fluid following S. pneumoniae challenge. A total cytokine induction score was calculated by summing the Z-score normalized to fold change for each of the measured cytokines. Paired volunteer numbers to baseline are indicated per time point after exposure (same numbers in Fig. 4B). Saliva clearers showed statistically significant total induction score of cytokines at 24 h postexposure compared to those of nasal clearers and culture-positives.