Hepatitis C virus (HCV) is a major public health concern, with over 70 million people infected worldwide, who are at risk for developing life-threatening liver disease. No vaccine is available, and immunity against the virus is not well-understood. Following the acute stage, HCV usually causes chronic infections. However, ~30% of infected individuals spontaneously clear the virus. Therefore, using HCV as a model for comparing immune responses between spontaneous clearer (SC) and chronically infected (CI) individuals may empower the identification of mechanisms governing viral infection outcomes. Here, we provide the first in-depth analysis of adaptive immune receptor repertoires in individuals with current or past HCV infection. We demonstrate that SC individuals, in contrast to CI patients, develop clusters of antibodies with distinct properties. These antibodies’ characteristics were used in a machine learning framework to accurately predict infection outcome. Using combinatorial antibody phage display library technology, we identified HCV-specific antibody sequences. By integrating these data with the repertoire analysis, we constructed two antibodies characterized by high neutralization breadth, which are associated with clearance. This study provides insight into the nature of effective immune response against HCV and demonstrates an innovative approach for constructing antibodies correlating with successful infection clearance. It may have clinical implications for prognosis of the future status of infection, and the design of effective immunotherapies and a vaccine for HCV.
HCV infection can lead to hepatitis, cirrhosis, liver failure, and hepatocellular carcinoma (HCC); it is the leading cause of liver transplantation (1). HCC is the fifth most common cancer, and the third leading cause of cancer-related death worldwide. Unfortunately, its prevalence in the US and Western Europe is increasing (1). No vaccine is currently available for HCV, and immunity against the virus is not well-understood. Cure rates are expected to increase with the recent approval of Direct-Acting Antiviral Drugs (DAAs). Yet, despite this progress, many challenges remain, such as limited implementation, efficacy, and protection from reinfection (2). Thus, global eradication of HCV by implementing DAAs is currently not a feasible goal (3–6). Since vaccination is considered the most effective means of eradicating viral infections (5), a prophylactic HCV vaccine is an urgent, unmet medical need (3–6). However, critical gaps in understanding the correlates of protective HCV immunity have hindered the design of anti-HCV vaccines and novel immunotherapeutics (3–6).
Unlike HIV-infections, which are not spontaneously cleared, 20–40% of HCV-infected individuals experience spontaneous recovery (7). A multitude of evidence suggests that induction of an efficient HCV-specific natural immunity can control the infection. Therefore, using HCV as a model for comparing immune responses between spontaneous clearer (SC) and chronically infected (CI) individuals will enable the identification of unique mechanisms that govern human disease outcomes. Until recently, protection against persistent HCV infection was thought to be associated with a vigorous T-cell response (8). However, it is now widely accepted that neutralizing antibodies (nAbs) also play a key role in viral clearance (8–12). This point was strengthened by demonstrating that natural clearance correlates with the early development of nAbs (13), and with nAbs that exhibit distinct epitope specificity (14). Extensive characterization of monoclonal HCV-neutralizing antibodies (mnAbs), combined with crystal structures of the HCV envelope protein E2, which is the target of most HCV-nAbs, has provided valuable information regarding the E2 antigenic landscape (15–19). However, since most HCV mnAbs characterized to date were generated from CI patients (12, 20, 21), the nature and epitope specificities of mnAbs in SC individuals remain to be elucidated. Recent studies have demonstrated that the early appearance of broadly neutralizing antibodies (bnAbs) is associated with spontaneous clearance (13). Interestingly, bnAbs also protect against HCV infection in animal models (22–24). Very recently, the first panels of bnAbs isolated from SC infections have been developed (25, 26). The panel reported by Bailey et al. displayed a low number of somatic mutations compared with the well-characterized nAbs from chronic patients exhibiting higher neutralization breadth, but were similar to nAbs from chronic infections in terms of clonality and epitope specificities (26). It remains unknown whether and how the immune response of SC individuals is distinct from that of CI patients.
New emerging technologies empowering high-throughput direct screening for specific antibodies have provided deep insights into the immunogens that elicit broad antibody responses (27, 28). In the case of HIV, such technologies led to the generation of broadly neutralizing monoclonal antibodies with significantly higher potency, breadth, and novel epitope specificities [reviewed in (29)]. These novel revolutionary methods of studying immune responses can offer important insights into the nature of immune responses to infections. The antibody repertoire of an individual stores information about current and past threats that the body has encountered, and thus has the potential to shed light on screening antibodies and vaccine design (27, 30). Comparing the features of antibody repertoires between distinct patient populations may provide information that can be correlated with clinically relevant outcomes (31, 32). Indeed, recent studies have found common antibody sequences in unrelated individuals following Dengue (33), influenza (34), and HIV (35) infections, as well as autoimmune diseases such as celiac (36) and pemphigus vulgaris (37). In chronic lymphocytic leukemia, 30% of patients carry highly similar antibodies (38). Here we utilized high-resolution technologies to identify unique antibodies that stratify between CI and SC HCV infection outcomes. We also used antibody repertoires in combination with phage display to construct HCV-specific broadly nAbs associated with HCV infection clearance.