![]() Serum was obtained 14, 38 and 83 days after CELDKWAS immunisation. The n-terminal C was added for efficient crosslinking to carrier and also included with the c-terminal S residue, part of the native sequence, to avoid problems with antibodies specific for just the terminal residue (see methods). The anti-ELDKWA response shows affinity maturationĮight mice were immunised with 50 ug of CELDKWAS peptide conjugated to KLH in alum, for each time-point. Determining where on this scale of specificity in vivo responses lie, and how they change as the response progresses, is an aim of the study reported here. ![]() With perfect specificity, an antibody response would not show binding to any epitope variants with no specificity, a response would show binding to all possible non-self epitopes. This issue is important, however, because it has a bearing on the related subjects of how broad the antibody reactivity induced by vaccine epitopes is, and similarly, the degree to which antibody responses to pathogen epitopes protect against variants of the epitope. Although generation of frank self-reactivity can lead to rapid apoptosis in germinal centres –, and so defines an extreme limit of the diversification of specificity, the overall scope of reactivity produced during antibody maturation is currently little understood. The genetic diversification caused by new V-gene recruitment and somatic hypermutation has, therefore, the potential to expand the number of epitopes bound by a developing antibody response. These different antibodies would each have slightly different antigen binding regions that may have specificity beyond that for the immunizing epitope, and there is also evidence that somatic mutation can change antibody specificity altogether. As an antibody response progresses the number of V-regions or CDR3s used against some antigens may reduce, although against others numbers may increase, and therefore while at a coarse level the repertoire may become more restricted, somatic hypermutation in germinal centers diversifies V-genes to such an extent that at the sequence level many cells have different receptors, , –. The serum response to T-dependent antigens is, however, a composite of antibodies from many clones, and it has long been known that ‘specific’ antibody responses produce antibodies that react with a variety of unrelated antigens. A single high affinity antibody might only bind one or a few targets with strength. It is often assumed that as antibodies increase in affinity they also become more specific, and at a trivial level this is likely. The relationship between serum antibody affinity and specificity is poorly understood. This implies that many pathogen epitope escape variants that could manifest as single amino-acid substitutions would not emerge by escaping immune surveillance. ![]() Thus, maturation of the antibody response to a single epitope results in a broadening of the high-affinity response toward variant epitopes. Furthermore there is a bias towards high affinity serum IgG binding to variant epitopes with conservative substitutions, although underlying this trend there is also significant binding to many epitopes with non-conservative substitutions. The serum IgG response is shown to mature and increase affinity for ELDKWA, and the titre and affinity of IgG against most epitope variants tested increases. To investigate this, a library of single amino-acid substitution epitope variants has been screened with serum obtained at different time-points after immunization of mice with the HIV gp41 peptide epitope ELDKWA. ![]() The effect this process has on the specificity of antibody for variants of the antigen is not well-defined, despite the potential role of antibody diversification in generating enhanced protection against pathogen escape mutants, or novel specificities after vaccination. During maturing antibody responses the increase in affinity for target antigens is achieved by genetic diversification of antibody genes followed by selection for improved binding.
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