Reverse vaccinology 2.0: Human immunology instructs vaccine antigen design

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

an in vivo RSV infection model in cotton rats (Kwakkenbos et al., 2010) . In a subsequent study, it was shown that D25 and two additional highly potent RSV-specific NAbs were specific for the prefusion form of RSV F (McLellan et al., 2013b) . Furthermore, by analyzing many different mAbs isolated from an RSV sero-positive individual, Corti et al. (2013) have also shown that the most potent NAbs recognize the prefusion form of the F antigen, suggesting that the prefusion protein could be considered as the most effective vaccine antigen. These examples of isolation and characterization of bNAbs induced by infection have highlighted how understanding the mechanisms leading to the elicitation of a protective response can aid the design of more effective vaccines. However, the identification of mAbs of the desired specificity usually requires the interrogation of thousands of B cells, with labor-intensive culture and screening steps of the B cell culture supernatants that lead to the isolation of a few dozen Abs. Moreover, this approach does not allow the gain of substantial information on the shaping of the Ig gene repertoire induced by the response to a given pathogen or vaccine. A milestone in understanding the nature and generation of successful Ab responses has been the development of Ab repertoire sequencing technologies for the identification and generation of recombinant human mAbs. The first description of the single-cell RT-PCR method showed the possibility of isolating the Ig heavy (VH) and light (VL) variable chain genes from single B cells, followed by the expression of chimeric murine and rabbit mAbs (Babcook et al., 1996) . Another hallmark in the field of human immunology has been the demonstration that cloning and expression of human mAbs could be achieved through the recovery of the Ig sequences from single B cells derived from the bone marrow and blood of healthy donors and by transfection of producer cells with the VH and VL chain genes (Wardemann et al., 2003) . Furthermore, the possibility of using fluorescently labeled antigens to sort antigen-specific B cells by flow cytometry has facilitated more focused analyses of the Ig gene repertoire usage and of the corresponding Ab binding and/or functional capability toward targeted antigen-specific and often rare B cell subpopulations after vaccination or infection (Tiller et al., 2008; Wrammert et al., 2008; Scheid et al., 2009a) . Although this methodology allows for sequencing and expression of the Abs encoded by a small number of B cells and individuals, it has brought numerous immunological insights to the development of the B cell response to antigens of a different nature. For example, it has been demonstrated that the B cell responses to polysaccharides, such as Streptococcus pneumoniae polysaccharides, show high clonality with a limited Ig gene usage and can be different in diverse age groups (Kolibab et al., 2005; Smith et al., 2013) . Instead, greater repertoire diversity has been highlighted between different individuals and even within a single individual at different time points after vaccination with protein antigens, such as influenza hemagglutinin (HA) and tetanus toxoid (TT; Wrammert et al., 2008; Frölich et al., 2010) . However, it is mainly the research in the HIV field that has demonstrated how the characterization of bNAbs, isolated from immune donors, and the knowledge of their pathway of generation can provide new insights into how to induce them thro