The immune response against hepatitis C virus (HCV) is rarely effective at clearing the virus, resulting in ∼170 million chronic HCV infections worldwide. Here we report that ligation of an HCV receptor (CD81) inhibits natural killer (NK) cells. Cross-linking of CD81 by the major envelope protein of HCV (HCV-E2) or anti-CD81 antibodies blocks NK cell activation, cytokine production, cytotoxic granule release, and proliferation. This inhibitory effect was observed using both activated and resting NK cells. Conversely, on NK-like T cell clones, including those expressing NK cell inhibitory receptors, CD81 ligation delivered a costimulatory signal. Engagement of CD81 on NK cells blocks tyrosine phosphorylation through a mechanism which is distinct from the negative signaling pathways associated with NK cell inhibitory receptors for major histocompatibility complex class I. These results implicate HCV-E2–mediated inhibition of NK cells as an efficient HCV evasion strategy targeting the early antiviral activities of NK cells and allowing the virus to establish itself as a chronic infection.

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. Human B cell technologies to identify fu...

Infection with hepatitis C virus (HCV), a leading cause of chronic liver diseases, can associate with B lymphocyte proliferative disorders, such as mixed cryoglobulinemia and non-Hodgkin lymphoma. The major envelope protein of HCV (HCV-E2) binds, with high affinity CD81, a tetraspanin expressed on several cell types. Here, we show that engagement of CD81 on human B cells by a combination of HCV-E2 and an anti-CD81 mAb triggers the JNK pathway and leads to the preferential proliferation of the naïve (CD27 ؊ ) B cell subset. In parallel, we have found that B lymphocytes from the great majority of chronic hepatitis C patients are activated and that naïve cells display a higher level of activation markers than memory (CD27 ؉ ) B lymphocytes. Moreover, eradication of HCV infection by IFN therapy is associated with normalization of the activation-markers expression. We propose that CD81-mediated activation of B cells in vitro recapitulates the effects of HCV binding to B cell CD81 in vivo and that polyclonal proliferation of naïve B lymphocytes is a key initiating factor for the development of the HCV-associated B lymphocyte disorders.monoclonal antibody ͉ multimeric engagement ͉ B cell antigen receptor ͉ cryoglobulinemia H epatitis C virus (HCV) is a positive-stranded RNA virus of the Flaviviridae family (1). The HCV genome is 9.6 kb in length, with one large translational ORF encoding a single polyprotein, which is processed by host and viral proteases into at least three structural and seven nonstructural proteins with various enzymatic activities (1). Two heavily N-glycosylated proteins E1 and E2 are virion-envelope proteins and form heterodimers in vitro (2). An estimated 170 million individuals are infected with HCV worldwide (3). HCV infection is associated with the development of chronic hepatitis, cirrhosis, and hepatocellular carcinoma (4). B cell abnormalities, including cryoglobulinemia (5) and an increased risk of B cell non-Hodgkin lymphoma (6, 7), have been reported in a minority of HCV infections.Until very recently, it was not possible to grow HCV in cell culture, therefore studies of virus interaction with human cells have been surrogated by the assessment of binding and entry of HCV recombinant glycoproteins (8) or virus pseudotypes (9).We have previously reported that HCV-E2 protein binds with high affinity to the large extracellular loop of human CD81 (CD81-LEL) and that ''bona fide'' HCV particles bind human CD81 (10). Recently, it has been demonstrated that CD81 is required for entry and infection of human cells by in vitro-generated infectious HCV (11). CD81 is a widely distributed cell-surface tetraspanin that participates in different molecular complexes on various cell types, including B, T, and natural killer (NK) cells (12). On human B cells, CD81 is known to form a costimulatory complex with CD19 and CD21 (13,14) and that coligation of the B cell antigen receptor (BCR) with any of the components of this costimulatory complex lowers the threshold required for BCR-mediated B cell proliferat...

Adjuvants increase vaccine potency largely by activating innate immunity and promoting inflammation. Limiting the side effects of this inflammation is a major hurdle for adjuvant use in vaccines for humans. It has been difficult to improve on adjuvant safety because of a poor understanding of adjuvant mechanism and the empirical nature of adjuvant discovery and development historically. We describe new principles for the rational optimization of small-molecule immune potentiators (SMIPs) targeting Toll-like receptor 7 as adjuvants with a predicted increase in their therapeutic indices. Unlike traditional drugs, SMIP-based adjuvants need to have limited bioavailability and remain localized for optimal efficacy. These features also lead to temporally and spatially restricted inflammation that should decrease side effects. Through medicinal and formulation chemistry and extensive immunopharmacology, we show that in vivo potency can be increased with little to no systemic exposure, localized innate immune activation and short in vivo residence times of SMIP-based adjuvants. This work provides a systematic and generalizable approach to engineering small molecules for use as vaccine adjuvants.