Table 1.
Demographics of study participants.
Table 2.
Characteristics of Haemophilus influenzae type a clinical isolates.
Table 3.
Antigens used to absorb pooled human serum.
Table 4.
Concentrations of IgM and IgG antibody specific for Hia polysaccharide and titres of bactericidal antibody against Hia 08–191 (with exogenous complement) in adult First Nations and non-First Nations individuals.
Fig 1.
Sensitivity of Hia isolates 11–139 (A) and 14–61 (B) to serum bactericidal effect varies among individual sera. Bacteria were incubated for 1 hour at 37°C, 5% CO2 with 12 individual sera or heat inactivated sera at concentrations of 12.5%, 6.25% or 3.12% and then viable bacteria were enumerated. Bactericidal activity was determined by comparing the number of viable bacteria exposed to serum versus heat inactivated serum. Mean percentage bactericidal activity is indicated. The graphs are representative of at least two independent experiments, each performed in triplicate, which gave similar results.
Fig 2.
Hia isolates 11–139 (A) and 14–61 (B) are sensitive to serum in a dose dependent manner. Bacteria were incubated for 1 hour at 37°C, 5% CO2 with pooled serum or heat inactivated serum at concentrations of 12.5%, 6.25% or 3.12% and then viable bacteria were enumerated. Bactericidal activity was determined by comparing the number of viable bacteria exposed to serum versus heat inactivated serum. The graphs are representative of three independent experiments, each performed in triplicate, which gave similar results.
Fig 3.
Hia serum sensitivity following Hia polysaccharide (PS) absorption.
Absorption of anti-Hia PS antibodies from pooled serum reduced serum sensitivity of Hia isolates. Isolates were incubated with mock-absorbed or Hia PS absorbed sera (12.5%) for 1 hour at 37o C and then viable bacteria were enumerated. Bars indicate mean percent bacterial killing determined by comparing viable bacteria following serum treatment versus heat inactivated serum ±SEM. Statistical significance was determined using Student's two tailed t-test. *P<0.05; ** P<0.01; ***P<0.001; (n.s.), not significant. The graph is representative of three independent experiments, each performed in triplicate, which gave similar results.
Fig 4.
Serum sensitivity of Hia isolates following nullification of the classical complement pathway: The majority of the serum bactericidal activity is antibody mediated (classical complement pathway).
Pooled human serum was mock or Mg2+EGTA treated and evaluated for bactericidal activity against select Hia isolates. Bars indicate mean percent bactericidal activity compared to heat inactivated sera ±SEM. Statistical significance was determined using Student's two tailed t-test. *P<0.05, ** P<0.01, ***P<0.001. The graph is representative of two independent experiments, each performed in triplicate, which gave similar results.
Fig 5.
Bactericidal activity of serum following absorption with different antigens: Anti-Hia LOS antibodies are a significant source of serum bactericidal activity against some Hia isolates.
The isolates 04–001 (A) and 11–173 (B) were incubated with mock or antigen absorbed pooled human serum. Human serum albumin was used as an irrelevant antigen. Bars indicate mean percent bacterial activity relative to mock absorbed sera ±SEM. Statistical significance was determined using one-way ANOVA with Dunnett's multiple comparison post hoc test. *P<0.05, ** P<0.01, ***P<0.001, HSA = human serum albumin, HiB PS = H. influenzae type b polysaccharide, 6B PS = Pneumococcal polysaccharide 6B, LOS = Hia lipooligosaccharide, LOS+Hia PS = LOS + Hia polysaccharide. The graphs are representative of three independent experiments, each performed in triplicate, which gave similar results.