Reverse vaccinology 2.0: Human immunology instructs vaccine antigen design

Rappuoli, Rino; Bottomley, Matthew J.; D’Oro, Ugo; Finco, Oretta; De Gregorio, Ennio

Overall, the single B cell cloning approaches used to isolate mAbs have been demonstrated to be extremely powerful for the identification and characterization of key protective antigens but have the drawback of being low-throughput technologies, revealing only a very limited slice of the full Ab repertoire. Therefore, using these techniques to analyze the impact that a vaccine antigen or a vaccine formulation can have in inducing specific repertoire signatures shared by different individuals may lead to erroneous and misleading conclusions. Recent advances in next-generation sequencing technology have enabled the sequencing of Ig genes from millions of B cells simultaneously, giving a high-resolution characterization of the Ab sequence repertoire (Reddy and Georgiou, 2011) . Nevertheless, the ability of this approach to recognize the antigen-specific Ab repertoire inside the totality of the individual Ab repertoire and evaluate specific changes induced by a vaccine or pathogen still remains questionable. One approach is to computationally search the total repertoire data for Ab sequences that are shared among individuals that have been exposed to the same antigen through infection or vaccination, identifying a "convergent repertoire." Three recent studies found that conserved CDR3 sequences were presence of feeder cells allow direct screening and selection of naturally produced Abs with desired functionality and recovery of the corresponding Ig gene sequence. This approach is one of the most recent that allows for the interrogation of single-sorted B cells through direct screening of Ab functionality. In turn, repertoire analyses to understand the origin and evolution of the Abs of interest can be performed . Starting from the recovered sequences, further expression of the recombinant Abs of interest in the most appropriate system allows for a fine-tuned characterization of their properties. (Center right) Structural characterization of such Abs bound to their target antigen (Ag) allows for the detailed definition of the protective epitope. A molecular detail of an antigen-Ab (Fab) complex as revealed by a cocrystal structure identifying a protective epitope is represented as an example. The protective epitope (red shape) can then be engineered for presentation as an optimized immunogen in a novel format (for example, by mounting the epitope in an oriented multicopy array on a nanoparticle [orange octagon], because nanoparticles can increase an epitope-focused immune response; López-Sagaseta et al., 2016) . (Bottom inset) The new antigen can be developed with the best formulation or delivery system to then be tested in humans. The figure was inspired by Fig. 3 from an earlier paper (Burton, 2002) . produced in patients recovering from acute dengue infection (Parameswaran et al., 2013) , or during the immune response to pandemic influenza H1N1 vaccination (Jackson et al., 2014) and Hib-polysaccharide vaccination (Trück et al., 2015) . Although the "convergent repertoire" demonstrates changes in the large-scale structural features of the repertoire, the antigen specificity of the identified Ab sequences always needs experimental confirmation through the expression, purification, and functional testing of recombinant Abs. However, only a small subset of Abs are usually expressed and investigated after the exhaustive interrogation of the repertoire, missing the possibility of having a full picture of the properties and functionality of all