Protein engineering strategies for rational immunogen design

Abstract

Antibody immunodominance refers to the preferential and asymmetric elicitation of antibodies against specific epitopes on a complex protein antigen. Traditional vaccination approaches for rapidly evolving pathogens have had limited success in part because of this phenomenon, as elicited antibodies preferentially target highly variable regions of antigens, and thus do not confer long lasting protection. While antibodies targeting functionally conserved epitopes have the potential to be broadly protective, they often make up a minority of the overall repertoire. Here, we discuss recent protein engineering strategies used to favorably alter patterns of immunodominance, and selectively focus antibody responses toward broadly protective epitopes in the pursuit of next-generation vaccines for rapidly evolving pathogens.

Fig. 1. Modulating precursor repertoire with various immunogen design strategies.

Schematic representation of how precursor frequency may be altered using different protein engineering strategies to remove ‘off-target’ competing clones using influenza hemagglutinin (HA) as a representative example. Colors refer to theoretical epitopes on full length HA; shades of blue and purple correspond to head-directed epitopes; shades of yellow, orange, and red correspond to stem-directed epitopes. For orthogonal grafting, a hypothetical scaffold is shown in gray with purple region illustrating a grafted HA head epitope. All images created in PyMol using PDB 5UGY.

Fig. 2. Design strategies to enhance ‘on-target’ epitope responses.

a Stem- and head-only HAs indicating locations of engineered prolines (left, red spheres) and inter-HA cysteines (right, yellow spheres) to stabilize the prefusion conformation. b Selective removal of native glycans to expose neoepitopes (red circle). c Computational design of HA antigens based on overall subtype diversity increases cross-reactive responses. To illustrate the COBRA approach, HA is arbitrarily colored in gray shading to show variation in amino acid identity that ultimately contributes to the consensus sequence; for a complete description see Huang et al.. d Optimization of a single epitope for a specific class of B cell precursors increases initial antigen affinity. HA receptor binding site (RBS) is shown in green as an example. e Resurfacing of a complex conformational-specific epitope allows for heterologous boosting of subdominant responses within memory. S1–S4 segments of the grafted RBS shown in red, orange, green, and blue, respectively. f Chimeric HAs where head domain (purple) of an antigenically distinct non-circulating HA is transplanted onto a conserved circulating stem domain (gray) to preferentially target stem-directed responses. All images created in PyMol using PDB 5UGY.

Fig. 3. Proposed use of heterologous antigen display to focus responses toward desired epitopes.

A proposed spectrum of epitope focusing immunogens incorporating various multimeric or heterologous display strategies is shown; unique colors represent antigenically distinct components. As the relative overall enrichment of the target epitope increases, so does the degree of epitope focusing. Heterologous display within a subtype presents a wider gradient of conserved epitopes due to the smaller antigenic distance between presented components. Heterologous display of antigenically distinct antigens enriches for conserved epitopes, but there are still other epitopes that are conserved between various combinations of component antigens. The endpoint of this spectrum is an epitope-enriched immunogen, where a single epitope is presented as multiple copies with all other epitopes presented once. All images created in PyMol using PDBs 5UGY and 3BVE.