TY - JOUR

T1 - Epitope Mapping of Polyclonal Antibodies by Hydrogen–Deuterium Exchange Mass Spectrometry (HDX-MS)

AU - Ständer, Susanne

AU - Grauslund, Laura R.

AU - Scarselli, Maria

AU - Norais, Nathalie

AU - Rand, Kasper

PY - 2021

Y1 - 2021

N2 - Epitope mapping of antibodies (Abs) is crucial for understanding adaptive immunity, as well as studying the mode of action of therapeutic antibodies and vaccines. Especially insights into the binding of the entire polyclonal antibody population (pAb) raised upon vaccination would be of unique value to vaccine development. However, very few methods for epitope mapping can tolerate the complexity of a pAb sample. Here we show how hydrogen–deuterium exchange mass spectrometry (HDX-MS) can be used to map epitopes recognized by pAb samples. Our approach involves measuring the HDX of the antigen in absence or presence of varied amounts of pAbs, as well as dissociating additives. We apply the HDX-MS workflow to pAbs isolated from rabbit immunized with factor H-binding protein (fHbp), a Neisseria meningitidis vaccine antigen. We identify four immunogenic regions located on the N- and C-terminal region of fHbp and provide insights into the relative abundance and avidity of epitope binding Abs present in the sample. Overall, our results show that HDX-MS can provide a unique and relatively fast method for revealing the binding impact of the entire set of pAbs present in blood samples after vaccination. Such information provides a rare view into effective immunity and can guide the design of improved vaccines against viruses or bacteria.

AB - Epitope mapping of antibodies (Abs) is crucial for understanding adaptive immunity, as well as studying the mode of action of therapeutic antibodies and vaccines. Especially insights into the binding of the entire polyclonal antibody population (pAb) raised upon vaccination would be of unique value to vaccine development. However, very few methods for epitope mapping can tolerate the complexity of a pAb sample. Here we show how hydrogen–deuterium exchange mass spectrometry (HDX-MS) can be used to map epitopes recognized by pAb samples. Our approach involves measuring the HDX of the antigen in absence or presence of varied amounts of pAbs, as well as dissociating additives. We apply the HDX-MS workflow to pAbs isolated from rabbit immunized with factor H-binding protein (fHbp), a Neisseria meningitidis vaccine antigen. We identify four immunogenic regions located on the N- and C-terminal region of fHbp and provide insights into the relative abundance and avidity of epitope binding Abs present in the sample. Overall, our results show that HDX-MS can provide a unique and relatively fast method for revealing the binding impact of the entire set of pAbs present in blood samples after vaccination. Such information provides a rare view into effective immunity and can guide the design of improved vaccines against viruses or bacteria.

U2 - 10.1021/acs.analchem.1c00696

DO - 10.1021/acs.analchem.1c00696

M3 - Journal article

C2 - 34308633

VL - 93

SP - 11669

EP - 11678

JO - Analytical Chemistry

JF - Analytical Chemistry

SN - 0974-7419

IS - 34

ER -