Reverse vaccinology 2.0: Human immunology instructs vaccine antigen design

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

Both of the aforementioned methods have two limitations for the identification of the best protective Abs: first, the required EBV transformation of B cells can have a bias on the secretion of mAbs; and second, in vitro-expressed synthetic Abs might not always faithfully reflect their natural functional capability. To overcome these limits, Burtion, Poignard, and co-workers have isolated HIV-specific human mAbs from MBCs expanded in vitro with the addition of feeder cells and conditioned medium generated from mitogen-stimulated human T cells and screened the supernatants for neutralization activity (Walker et al., 2009 (Walker et al., , 2011 . A modified high-throughput approach has also recently been developed based on the isolation of MBCs from PBMCs and expansion in vitro without the need for activated T cell supernatants, allowing secretion of sufficient amounts of Abs in culture to be screened in binding and functional assays . The main advantage of this technique is provided by the possibility of isolating and directly characterizing the Abs produced by individually cloned B cells without the need for cloning and expression of all the recombinant mAbs and, more importantly, maintaining the original natural properties of binding and functionality. After characterization of the binding and/or functional capacities of the Abs, the Ig gene sequences can be recovered. In turn, repertoire analyses to understand the origin and evolution of the Abs of interest can be performed, and further cloning and expression steps for deeper structural characterizations can be performed (Fig. 1) . Recently, through this new approach, broad and potent HIV NAbs have been discovered in the pool of antigen-specific MBCs, highlighting new vulnerability sites on the HIV Env antigen (Huang et al., 2014) . This efficient new methodology, applicable both to preselected antigen-specific single-sorted MBCs or to B cell subpopulations of unknown specificity, will enable the identification of new target antigens or epitopes involved in a protective Ab response (Fig. 1) .