Human Infant Memory B Cell and CD4+ T Cell Responses to HibMenCY-TT Glyco-Conjugate Vaccine.

Carrier-specific T cell and polysaccharide-specific B cell memory responses are not well characterised in infants following glyco-conjugate vaccination. We aimed to determine if the number of Meningococcal (Men) C- and Y- specific memory B cells and; number and quality of Tetanus Toxoid (TT) carrier-specific memory CD4+ T cells are associated with polysaccharide-specific IgG post HibMenCY-TT vaccination. Healthy infants received HibMenCY-TT vaccine at 2, 4 and 6 months with a booster at 12 months. Peripheral blood mononuclear cells were isolated and polysaccharide-specific memory B cells enumerated using ELISpot. TT-specific memory CD4+ T cells were detected and phenotyped based on CD154 expression and intracellular TNF-α, IL-2 and IFN-γ expression following stimulation. Functional polysaccharide-specific IgG titres were measured using the serum bactericidal activity (SBA) assay. Polysaccharide-specific Men C- but not Men Y- specific memory B cell frequencies pre-boost (12 months) were significantly associated with post-boost (13 months) SBA titres. Regression analysis showed no association between memory B cell frequencies post-priming (at 6 or 7 months) and SBA at 12 months or 13 months. TT-specific CD4+ T cells were detected at frequencies between 0.001 and 0.112 as a percentage of CD3+ T cells, but their numbers were not associated with SBA titres. There were significant negative associations between SBA titres at M13 and cytokine expression at M7 and M12.Induction of persistent polysaccharide-specific memory B cells prior to boosting is an important determinant of secondary IgG responses in infants. However, polysaccharide-specific functional IgG responses appear to be independent of the number and quality of circulating carrier-specific CD4+ T cells after priming

Men C- and Men Y- specific memory B cell responses following HibMenCY-TT vaccine.

<p><b>A.</b> Men C- and <b>B.</b> Men Y- specific Antibody Forming Cells (AFCs) per 1x10⁶ cultured PBMCs after 2 (M6) or 3 (M7) priming doses, and prior to (M12) and 1 month post (M13) the booster dose of HibMenCY-TT, where each point represents the response from one individual and horizontal bars indicate median values. Individuals with no detectable spots were given an arbitrary value of 1. Red dots represent paired samples from individuals who had blood taken following 2 doses of HibMenCY-TT prime, whilst black dots represent those samples from individuals who had blood taken after 3 doses of HibMenCY-TT prime. The dashed line represents the assay limit of detection. The numbers below the x axis of each graph indicate the percentage of subjects with detectable memory B cells specific for that antigen at that time point. p(**)≤0.01 and p(*)≤0.05 indicating significance of independent samples Mann-Whitney U tests or related samples Wilcoxon signed rank tests.</p

Men C- and Men Y- specific SBA titres following HibMen CY-TT vaccine.

<p><b>A.</b> Men C- and <b>B.</b> Men Y- specific SBA titre after 2 (M6) or 3 (M7) priming doses, and prior to (M12) and 1 month post (M13) the booster dose of HibMenCY-TT vaccine. Red dots represent paired samples from individuals who had blood taken following 2 doses of HibMenCY-TT prime, whilst black dots represent those samples from individuals who had blood taken after 3 doses of HibMenCY-TT prime. Horizontal bars indicate median values. The dashed line represents the assay limit of detection. p(**)≤0.01 and p(*)≤0.05 indicating significance of independent samples Mann-Whitney U tests or related samples Wilcoxon signed rank tests.</p

Single and double positive TT-specific memory CD4+ T cell cytokine responses following HibMenCY-TT vaccine.

<p>Intracellular TNFα, IFNγ, and IL-2 production by TT-specific memory cells was determined 48 hours after stimulation with TT, in PBMCs from infants after 2 doses (M6) or 3 doses (M7) of prime, and prior to (M12), and 1 month post (M13) a booster dose of HibMenCY-TT. <b>A.</b> single-positive TNFα+, IFNγ+ and IL-2+ cells and <b>B.</b> double-positive TNFα+IFNγ+, IL-2+IFNγ+ and TNFα+IL-2+ cells. Mean + SEM are shown. p(*)≤0.05 indicating significance of related samples Wilcoxon signed rank tests.</p

Association of Men C- and Men Y- specific memory B cells with SBA titre pre- and post- boost.

<p>Men C-specific memory B cells at M6 and M7 were plotted against log-transformed M12 Men C SBA (A and B) and M13 SBA (C and D). M12 Men C-specific memory B cells were plotted against log-transformed M13 SBA values (E). Men Y-specific memory B cells at M6 and M7 were plotted against log-transformed M12 Men Y SBA (F and G) and M13 SBA (H and I). M12 Men Y-specific memory B cells were plotted against log-transformed M13 SBA values (J). Linear regression analyses were performed to determine statistically significant associations where r = r² value, n = number of subjects and p(*)≤0.05.</p

Association of TT-specific memory T cell responses with SBA titre pre- and post- boost.

<p>TT-specific CD4+CD154+ T cells at M6 and M7 were plotted against log-transformed M12 Men C SBA (A and B) and M13 SBA (C and D). M12 TT-specific CD4+CD154+ T cells were plotted against log-transformed M13 SBA values (E). TT-specific CD4+CD154+ T cells at M6 and M7 were plotted against log-transformed M12 Men Y SBA (F and G) and M13 SBA (H and I). M12 TT-specific CD4+CD154+ T cells were plotted against log-transformed M13 SBA values (J). Linear regression analyses were performed to determine statistically significant associations where r = r² value and n = number of subjects.</p

TT-specific CD4+CD154+ CD3+ responses over time following HibMenCY-TT vaccine.

<p>The frequency above background (Δ) of TT-specific CD4+CD154+ memory cells in CD3+ lymphocytes was determined in PBMCs from infants after 2 doses (M6) or 3 doses (M7) of prime, prior to (M12), and 1 month post (M13) a booster dose of HibMenCY-TT. Each point represents the response from 1 individual and the horizontal bars represent median values. Red dots represent paired samples from individuals who had blood taken following 2 doses of HibMenCY-TT prime, whilst black dots represent those samples from individuals who had blood taken after 3 doses of HibMenCY-TT prime. p(*)≤0.05, indicating significance of related samples Wilcoxon signed rank tests.</p

High concentrations of middle ear antimicrobial peptides and proteins and proinflammatory cytokines are associated with detection of middle ear pathogens in children with recurrent acute otitis media.

Recurrent and chronic otitis media (OM) are often refractory to antibiotics due to bacterial persistence in biofilm within the middle ear. In vitro and in vivo studies have demonstrated that antimicrobial proteins and peptides (AMPs) are bactericidal against otopathogens, indicating potential therapeutic value for recalcitrant OM. We measured concentrations of 6 AMPs and 14 cytokines in middle ear effusion (MEE) from 67 children undergoing ventilation tube insertion for recurrent acute OM. Sixty one percent of children had bacterial otopathogens detected in their MEE, 39% by PCR and 22% by PCR and culture. Groups were defined as: PCR-negative/culture-negative (absence of bacterial otopathogen), n = 26; PCR-positive/culture-negative (presence of nonculturable bacterial otopathogen), n = 26; PCR-positive/culture-positive (presence of culturable bacterial otopathogen), n = 15. Age, antibiotic usage, day-care attendance, presence of respiratory viruses in MEE and number of AOM episodes were similar between groups. AMP and cytokine concentrations were higher in children with bacterial otopathogens in their MEE compared to those with no bacterial otopathogens. Median concentrations of AMPs (except HBD2) were 3 to 56-fold higher in MEE from children with bacterial otopathogens detected in their MEE (P ≤ 0.01). Similarly, median cytokine concentrations (except TGFβ) were >16-fold higher in MEE with bacterial otopathogens detected (P ≤ 0.001). This is the first study to measure AMPs in MEE and together with the cytokine data, results suggest that elevated AMPs and cytokines in MEE are a marker of inflammation and bacterial persistence. AMPs may play an important role in OM pathogenesis