Abstract by Laura Rokkedahl Grauslund

Antibodies play a crucial role in the defense against infectious pathogens and in the immune response after vaccination. The fundamental understanding of the antigen-antibody interaction is crucial for the development of efficient vaccines. Advanced analytical techniques are required to gain detailed information about the epitopes recognized by the antibodies produced after vaccination. In this PhD project, the humoral immune response to vaccination was investigated through the use of hydrogen/deuterium exchange mass spectrometry (HDX-MS). Antigens from a vaccine against Neisseria meningitidis serogroup B (MenB) and antibodies raised against the vaccine antigens, have been used for epitope mapping studies. MenB is a gram-negative bacterium that causes severe meningococcal disease across the world.

In the first part, two monoclonal antibodies (mAbs) specific for the antigen Neisserial adhesin A (NadA) have been used for epitope and paratope mapping with HDX-MS in combination with surface plasmon resonance. Although the two mAbs bind to NadA with similar affinity, one mAb is able to provide protection against MenB alone, while the other mAb only provide protection against MenB in combination with other mAbs.

In the second part, a workflow for HDX-MS epitope mapping using polyclonal antibody (pAb) have been developed and optimized. Rabbits immunized with the antigen factor H binding protein (fHbp) have been used as source for extraction of pAb samples. The feasibility of the developed workflow have been demonstrated using pAb samples with and without enrichment of the fHbp-specifc antibodies. The results showed that the developed HDX-MS workflow allows identification of immunodominant epitopes across different pAb samples.

In the final part, the developed HDX-MS epitope mapping workflow was used for analysis of fHbp with human pAb samples from two vaccinated donors. The epitope mapping of fHbp was performed with and without enrichment of the fHbp-specific antibodies. The HDX-MS epitope mapping enabled the identification of immunodominant epitopes recognized by the pAbs from both vaccinated donors. The results showed that human pAb samples are feasible for use in HDX- MS epitope mapping without the necessary requirement of enrichment of antigen-specific antibodies.