Development of a serum bactericidal antibody assay for Haemophilus influenzae type b

Townsend, Kelly (2013) Development of a serum bactericidal antibody assay for Haemophilus influenzae type b. Masters by Research thesis (MSc), Manchester Metropolitan University.

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Abstract

Prior to routine immunisation, Haemophilus influenzae serotype b (Hib) was a major cause of serious bacterial infections, particularly in young children. In 1992, immunisation against Hib disease was introduced into the infant vaccination schedule along with a catch-up campaign in those aged 1-4 years which resulted in a rapid and sustained reduction in invasive Hib disease across all age groups. Since 1999, however, the number of reported invasive Hib infections began to rise. In 2006, the routine infant immunisation schedule was changed to include a booster immunisation to Hib in the second year of life. This had an immediate effect, resulting in a rapid and sustained reduction in invasive Hib disease across all age groups. Evaluation of the immune response to Hib conjugate vaccines includes the measurement of serum antibodies to the Hib capsular polysaccharide (polyribosyl-ribitol-phosphate (PRP)) by ELISA, with accepted short term and long term levels of ≥0.15 μg/mL and ≥1.0 μg/mL, respectively. The relevance for protection in children who have been primed with glycoconjugate vaccines remains unclear, as these levels were derived by passive immunisation, or immunisation with pure polysaccharide. Therefore the aim of this project was to develop and optimise a serum bactericidal antibody (SBA) assay for the evaluation of Hib conjugate vaccines. In order to assess the functional activity of Hib antibodies, we developed, optimised and evaluated a serum bactericidal antibody (SBA) assay. Validation of the Hib SBA assay was deemed acceptable in all assay parameters tested. In vaccinated adults, a strong correlation (r=0.81) between anti-PRP IgG concentrations and SBA titres were shown. The optimised Hib SBA assay was tested on sera from infants immunised under the current UK 2-3-4-12 month schedule. Good correlations between anti-PRP IgG concentration and SBA 16 titres were shown. (r=0.635, post primary, r=0.746, post booster). A predictive SBA titre of 8 was calculated using the established correlates of protection. As Hib strains may contain multiple capsular loci, we also performed the SBA assay with strains expressing 1-5 copies of the cap b locus using convalescent sera from children with Hib vaccine failure. Geometric mean SBA titres for 1, 2, 3, 4 and 5 copy strains were 46, 24, 43, 55 and 20, respectively. There was no strong association between the number of cap b copies and SBA titres. Although an increased capsule may be an advantage, antibodies to other surface antigens may play a role in clearance.