With the development of monovalent serogroup A and tetravalent serogroup A, C, W135 and Y conjugate vaccines, attention is now focused on the use of serological assays for correlates of vaccine-induced protection, particularly for serogroup A. The main aim of this project was to further understand the immune response to meningococcal capsular polysaccharides with an emphasis on serogroup A. Due to concerns over the most appropriate complement source to use in the serum bactericidal antibody (SBA) assay, a comparison between human and rabbit complement was performed, using three different target strains. The degree of serogroup A capsular polysaccharide expression and the immunotype of the three strains were determined. Serum bactericidal activity which was high in the presence of rabbit complement, was abolished when human complement was used. Factor H was not the cause of this inhibition. An enzyme-linked immunosorbent assay (ELISA) was developed to measure the level of vaccine-induced antibodies to meningococcal serogroup A polysaccharide (MenA). Several methods of attaching the antigen to microtitre plates were investigated and the use of methylated human serum albumin (MHSA) was found to be the most appropriate method. This method was used to investigate the antibody isotype response following vaccination. The predominant subclass following conjugate or polysaccharide vaccination was IgG with a greater proportion of IgM in those that received polysaccharide vaccine. Due to poor correlations between SBA titres and total IgG concentrations, methods for measuring or selecting for high avidity antibodies using ammonium thiocyanate were also investigated with the determination of avidity indices being the most appropriate. The avidity indices in an infant cohort following three doses of meningococcal serogroup A and C conjugate (MACC) vaccine were shown to increase progressively over time indicating avidity maturation. Determination of avidity indices in a toddler cohort which had received either MACC or meningococcal serogroup A and C polysaccharide (MACP) vaccine, showed that MenA polysaccharide may not behave as a classical T-cell independent antigen. Avidity indices in adults were shown to be less useful with no significant difference in the avidity indices of those that were vaccinated with either MACC or MACP. As multiple doses of meningococcal serogroup A polysaccharide vaccine have been recommended for protecting African children from disease the effect of this on serogroup A antibody levels was investigated. It was found that a second dose of MACP vaccine 6 months after primary vaccination elicited a reduced immunological response in young U. K. adults. Three international reference sera were quantified for meningococcal serogroup A, C, W135 and Y specific IgG subclass concentrations. Using the calibrated reference serum, the serogroup A-specific IgGi and IgG2 distribution following MenA vaccination in adults and infants was determined. The serogroup A-specific IgG response in infants was shown to be restricted to IgG1, whereas in adults both IgG1 and IgG2 were present. The serogroup A, C, W135 and Y specific IgGi and IgG2 responses following tetravalent A, C, W135 and Y conjugate vaccination in adults was shown to be heterogeneous with either IgG1 or IgG2 dominating the response. Assays were developed and optimised for the determination of the immune response to MenA polysaccharide and will be valuable for the future evaluation of meningococcal serogroup A vaccines. The information gained on the immune response to MenA polysaccharide adds to our knowledge and will assist in the understanding of the immune response to MenA polysaccharide.

The self-controlled case series (SeeS) method is commonly used to investigate associations between vaccine exposures and adverse events (side effects). It is an alternative to cohort and case control study designs. It requires information only on cases, individuals who have experienced the adverse event at least once, and automatically controls all fixed confounders that could modify the true association between exposure and adverse event. However, timevarying confounders (age, season) are not automatically controlled. The sees method has parametric and semi-parametric versions in terms of controlling the age effect. The parametric method uses piecewise constant functions with a priori chosen age ~ . groups and the semi-parametric method leaves the age effect unspecified. Mis-specification of age groups in the parametric version may lead to biased estimates of the exposure effect, and the semi-parametric approach runs into computational problems when the sample size is moderately large . Moreover, both versions of sees represent the time-varying exposures using step functions with pre-determined cut-points. A less prescriptive approach may be beneficial when the shape of the relative risk function associated with exposure is not known a priori, especially when exposure effects can be long-lasting. i :1 This thesis focuses on extending the sees method to avoid the aforementioned limitations I by modelling the age and exposure effects using flexible smooth functions. Specifically, we used penalised regression splines based on cubic M-splines, which are piecewise polynomials of degree 3. We developed three new extensions: a method that represents only the age effect with splines, a method that uses splines to model only the exposure effect and a non-parametric sees method that represents both effects by splines. Simulation studies showed that these new methods outperformed the parametric and semi-parametric methods. The new methods are illustrated using large data sets. Review of sees vaccine studies and directions on how to use the method are also given. "':-.' ' .

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