Table 1.

A toolkit for structural vaccinology

Property analyzed	Techniques	Utility
Three-dimensional structure of antigens and antigen-antibody complexes	X-ray crystallography, NMR, cryo-EM	Allow rational engineering by defining domain boundaries, epitope structure, and underlying architecture
Antigenic structure	ELISA, IP, escape mutant analysis, DXMS, phage display	Define the link between physical structure and the landscapes recognized by antibodies
Post-translational modification	SDS–PAGE, MS, glycosidic linkage analysis, X-ray crystallography, NMR	Assess the authenticity and homogeneity of modifications on recombinantly expressed proteins
Protein folding and stability	CD, ITC, DXMS, NMR, DSC, protease protection, native- and SDS–PAGE	Assess antigen conformation and integrity in solution over time for vaccine stability.
Non-covalent association and hydrodynamic radius	AUC, DLS, SEC, SPR	Assess antigen valency and aggregation

Selection of optimized protein antigens will require a comprehensive evaluation of the structural features of the recombinantly produced protein. Ideally, these analytical tools will be applied in a high throughput manner to many candidate structures. Abbreviations: AUC, analytical ultracentrifugation; CD, circular dichroism spectroscopy; cryo-EM, electron cryomicroscopy; DLS, dynamic light scattering; DSC, differential scanning calorimetry; DXMS, deuterium exchange mass spectrometry; ELISA, enzyme-linked immunosorbent assay; IP, immunoprecipitation; ITC, isothermal titration calorimetry; mAb, monoclonal antibody; MS, mass spectrometry; NMR, nuclear magnetic resonance spectroscopy; SEC, size exclusion chromatography. SPR, surface plasmon resonance;