Reverse vaccinology 2.0: Human immunology instructs vaccine antigen design

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

Traditionally, vaccines have been developed empirically by isolating, inactivating, and injecting the microorganisms (or portions of them) that cause disease (Table 1 ; Rappuoli, 2014) . Two decades ago, genome sequencing revolutionized this process, allowing for the discovery of novel vaccine antigens starting directly from genomic information. The process was named "reverse vaccinology" to underline that vaccine design was possible starting from sequence information without the need to grow pathogens (Rappuoli, 2000) . Indeed, a vaccine against meningococcus B, the first deriving from reverse vaccinology, has recently been licensed (Serruto et al., 2012; O'Ryan et al., 2014) . Today, a new wave of technologies in the fields of human immunology and structural biology provide the molecular information that allows for the discovery and design of vaccines against respiratory syncytial virus (RSV) and human CMV (HCMV) that have been impossible thus far and to propose universal vaccines to tackle influenza and HIV infections. Here, we provide our perspective (summarized in Table 1 ) of how several new advances, some of which have been partially discussed elsewhere (Burton, 2002; Dormitzer et al., 2012; Haynes et al., 2012) , can be synergized to become the engine driving what might be considered a new era in vaccinology, an era in which we perform "reverse vaccinology 2.0." Several technological breakthroughs over the past decade have potentiated vaccine design. First, the greatly enhanced ability to clone human B cells and then to produce the corresponding recombinant mAbs or antigen-binding fragments (Fab's) has provided access to an enormously rich set of reagents that allows for the proper evaluation of the protective human immune response to any given immunogen upon immunization or infection. A fundamental step for the success of this approach has been the growing capacity to select the most favorable donors for the isolation of the most potent antibodies (Abs) through extensive examination of serum-functional Ab responses. Second, conformational epitope mapping studies, performed via improved structural biology tools for the three-dimensional characterization of Fab's complexed with their target antigens , can now readily yield the atomic details of protective epitopes recognized by broadly neutralizing Abs (NAbs [bNAbs] ). Third, new computational approaches, informed by such structural and immunological data, have enabled the rational design of novel immunogens to specifically elicit a focused immune response targeting the most desirable protective epitopes (Liljeroos et al., 2015) . In addition to these advances, a great improvement in RNA sequencing technology has allowed for a massive analysis of the B cell repertoire, providing an accurate overview of the Ab maturation process generated by an infection or vaccination and driving new strategies aimed at priming the B cell precursors expressing germline-encoded Abs in an effective way before initiation of any somatic mutation.