Antibody responses targeting ApoA-I have been documented in patients suffering from chronic inflammation associated with obesity, autoimmunity and cardiovascular disease (CVD) and correlate with a poor prognosis. Patients presenting with an acute myocardial infarction (AMI) have measurable IgG targeting the lecithin:cholesterol acyltransferase (LCAT) domain of ApoA-I and are shown to bind an epitope containing an oxidized methionine (Met-148(O)). Antibodies targeting ApoA-I are thought to be serum markers for CVD and not responsible for disease progression, but their exact role is poorly understood. We hypothesize that the B cell response to ApoA-I is nuanced with different isotypes of both protective and pathologic antibodies induced during the course of disease. To investigate this hypothesis, we synthesized a series of unmodified and modified peptides corresponding to the LCAT domain of mouse, monkey and human ApoA-I for ELISA and immunization studies. ELISA with commercially available goat polyclonal antibodies induced by immunization with human ApoA-I suggest that the LCAT domain possesses immunogenic properties and the antibodies are capable of binding to the unmodified and Met-148(O) peptides. Although affinity to the unmodified peptide appears to be higher by ELISA, pre-incubating the polyclonal antibodies with a solution of the Met-148(O) peptide significantly inhibits binding to the unmodified peptide. These data suggest that the oxidized peptide in solution may undergo a structural change that increases antibody affinity. Structural analysis of the peptides are ongoing. Serum analysis from patients presenting with an AMI and 30 days after cardiac event show a decline in IgG responses toward the Met-148(O)-modified peptide during that period. Analysis of monkey serum provides a unique comparison. Monkeys have a methionine to valine mutation at position 148, which results in a non-oxidizable residue, and instead show a decrease in IgG toward the unmodified sequence when on an atherogenic diet for 12 weeks. Correlating these ELISA assays with atherosclerosis observed in the patients and macaques along with ongoing
in vivo
immunization studies in mice will generate insight into the nuanced B cell response toward ApoA-I during CVD.
Complete analysis of the B-cell response to a protein antigen, from in vivo germinal centre formation to 3-D modelling of affinity maturation