Learning from a clinical cohort for HCV vaccine development

About 71 million people worldwide suffer from chronic hepatitis C virus (HCV) infection. Without available treatment the large majority of HCV-infected patients progress to chronic disease. Chronic HCV infection is one of the most important risk factors for liver disease, including liver fibrosis, cirrhosis and hepatocellular carcinoma. Despite the development of efficient direct-acting antivirals with success rates of more than 95%, HCV incidence rates are still increasing, which is in part due to the ongoing opioid epidemic that is currently taking its toll in the US. High costs of treatment and the possibility for reinfection, which is frequent among intravenous drug users, warrant the development of a protective vaccine.[1]Baumert T.F. Fauvelle C. Chen D.Y. Lauer G.M. A prophylactic hepatitis C virus vaccine: a distant peak still worth climbing.J Hepatol. 2014; 61: S34-44Google Scholar The small but significant rate of spontaneous HCV clearance, in some rare cases even after years of chronic infection, suggests that that the development of a protective vaccine is not an impossible task. Despite large progress, the immune correlates of protection against infection are still only partially understood. Early vaccine studies involving chimpanzees[2]Choo Q.L. Kuo G. Ralston R. Weiner A. Chien D. Van Nest G. et al.Vaccination of chimpanzees against infection by the hepatitis C virus.PNAS. 1994; 91: 1294-1298Google Scholar have shown that long-lasting CD4 and CD8 T-cell responses play a significant role in HCV clearance, with loss of robust CD4+ response resulting in disease progression.[3]Gerlach J.T. Diepolder H.M. Jung M.C. Gruener N.H. Schraut W.W. Zachoval R. et al.Recurrence of hepatitis C virus after loss of virus-specific CD4(+) T-cell response in acute hepatitis C.Gastroenterology. 1999; 117: 933-941Google Scholar On the other side, B-cell responses and broadly neutralizing antibodies have been associated with HCV clearance.4Pestka J.M. Zeisel M.B. Blaser E. Schurmann P. Bartosch B. Cosset F.L. et al.Rapid induction of virus-neutralizing antibodies and viral clearance in a single-source outbreak of hepatitis C.PNAS. 2007; 104: 6025-6030Google Scholar, 5Osburn W.O. Fisher B.E. Dowd K.A. Urban G. Liu L. Ray S.C. et al.Spontaneous control of primary hepatitis C virus infection and immunity against persistent reinfection.Gastroenterology. 2010; 138: 315-324Google Scholar, 6de Jong Y.P. Dorner M. Mommersteeg M.C. Xiao J.W. Balazs A.B. Robbins J.B. et al.Broadly neutralizing antibodies abrogate established hepatitis C virus infection.Sci Transl Med. 2014; 6: 254ra129Google Scholar, 7Law M. Maruyama T. Lewis J. Giang E. Tarr A.W. Stamataki Z. et al.Broadly neutralizing antibodies protect against hepatitis C virus quasispecies challenge.Nat Med. 2008; 14: 25-27Google Scholar, 8Lavillette D. Morice Y. Germanidis G. Donot P. Soulier A. Pagkalos E. et al.Human serum facilitates hepatitis C virus infection, and neutralizing responses inversely correlate with viral replication kinetics at the acute phase of hepatitis C virus infection.J Virol. 2005; 79: 6023-6034Google Scholar Broadly neutralizing antibodies, meaning antibodies that neutralize more than 1 HCV genotype (GT) can be found in both patients with chronic HCV and in clearers. However, individuals clearing the infection were characterized by more rapid development of broadly neutralizing antibodies compared to those ultimately progressing to chronic HCV infection.[5]Osburn W.O. Fisher B.E. Dowd K.A. Urban G. Liu L. Ray S.C. et al.Spontaneous control of primary hepatitis C virus infection and immunity against persistent reinfection.Gastroenterology. 2010; 138: 315-324Google Scholar While a large number of patient-derived broadly neutralizing monoclonal antibodies effectively protect against or control an already established infection in liver humanized mice (reviewed in9Keck M.L. Wrensch F. Pierce B.G. Baumert T.F. Foung S.K.H. Mapping determinants of virus neutralization and viral escape for rational design of a hepatitis C virus vaccine.Front Immunol. 2018; 9: 1194Google Scholar, 10Keck Z.Y. Wang Y. Lau P. Lund G. Rangarajan S. Fauvelle C. et al.Affinity maturation of a broadly neutralizing human monoclonal antibody that prevents acute hepatitis C virus infection in mice.Hepatology. 2016; 64: 1922-1933Google Scholar), the development of a protective vaccine has been a major challenge. A prospective HCV vaccine does not necessarily need to provide complete sterilizing immunity, although this certainly would be the most desirable outcome. It might be sufficient to delay development of chronic infection to give the immune system a head start towards the elicitation of broadly neutralizing antibodies. This would appear to be similar to a spontaneous clearance of HCV infection and could be sufficient to help the host immune response stay ahead of the virus during the acute phase of HCV infection. This is further supported by the finding that spontaneous clearance rates increase from about 25% for a primary infection to 30 to 60% for those with 1 or multiple reinfections.[11]Sacks-Davis R. Grebely J. Dore G.J. Osburn W. Cox A.L. Rice T.M. et al.Hepatitis C virus reinfection and spontaneous clearance of reinfection–the InC3 study.J Infect Dis. 2015; 212: 1407-1419Google Scholar While several studies link the early development of neutralizing antibody responses with HCV clearance,4Pestka J.M. Zeisel M.B. Blaser E. Schurmann P. Bartosch B. Cosset F.L. et al.Rapid induction of virus-neutralizing antibodies and viral clearance in a single-source outbreak of hepatitis C.PNAS. 2007; 104: 6025-6030Google Scholar, 5Osburn W.O. Fisher B.E. Dowd K.A. Urban G. Liu L. Ray S.C. et al.Spontaneous control of primary hepatitis C virus infection and immunity against persistent reinfection.Gastroenterology. 2010; 138: 315-324Google Scholar the B-cell repertoire of individuals spontaneously clearing HCV infection and specific epitopes associated with protection have not yet been analyzed in detail. In that regard, individuals clearing multiple singular infections could be of particular interest as they could provide crucial information on correlates of protective immunity. Consequently, a thorough breakdown of B-cell responses associated with viral clearance will be paramount for HCV vaccine development. Merat and Bru et al. from AIMM Therapeutics and the University of Amsterdam aimed to shine a light on the role of the B-cell responses during spontaneous HCV clearance and long-term immunity.[12]Merat S.J. Bru C. van de Berg D. Molenkamp R. Tarr A.W. Koekkoek S. et al.Cross-genotype AR3-specific neutralizing antibodies confer long-term protection in injecting drug users after HCV clearance.J Hepatol. 2019; 71: 14-24Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar To perform a detailed analysis of the B-cell repertoire of patients with chronic HCV compared to spontaneous clearers, they took advantage of the long-term Amsterdam Cohort Study among injection drug users. Of the 13 individuals analyzed from the cohort, 5 were chronically infected, either after primary infection or reinfection, while the other 8 remained HCV negative after primary or after 1 or even multiple reinfections. By performing multiplex flow-cytometry analyzing antibody binding to HCV envelope glycoproteins E1E2 of different GTs, the authors revealed that antibodies isolated from clearers present a much broader reactivity than those isolated from chronically infected patients, which confirms previous studies.4Pestka J.M. Zeisel M.B. Blaser E. Schurmann P. Bartosch B. Cosset F.L. et al.Rapid induction of virus-neutralizing antibodies and viral clearance in a single-source outbreak of hepatitis C.PNAS. 2007; 104: 6025-6030Google Scholar, 5Osburn W.O. Fisher B.E. Dowd K.A. Urban G. Liu L. Ray S.C. et al.Spontaneous control of primary hepatitis C virus infection and immunity against persistent reinfection.Gastroenterology. 2010; 138: 315-324Google Scholar Out of 8 clearers, 7 had antibodies that showed reactivity against at least 2 HCV GTs and 5 of these had antibodies that showed cross-reactivity against at least 5 different GTs. Only 3 out of 5 chronically infected individuals showed reactivity against 2 or more GTs, with none of these reacting with more than 4 GTs. To obtain a more detailed understanding of the cross-neutralizing B-cell response in individuals clearing the infection, the authors analyzed the epitope specificity of antibodies secreted from B-cell cultures obtained from the 4 clearers with the highest frequency of cross-neutralizing antibodies. Most binding sites of broadly neutralizing antibodies cluster into 4 epitopes on the viral envelope protein designated as antigenic regions or domains (see ref[9]Keck M.L. Wrensch F. Pierce B.G. Baumert T.F. Foung S.K.H. Mapping determinants of virus neutralization and viral escape for rational design of a hepatitis C virus vaccine.Front Immunol. 2018; 9: 1194Google Scholar for a description of overlap between the AR and antigenic domain regions). Alanine mutant scanning of these 4 epitopes (epitope I/antigenic domain E, epitope II/antigenic domain D, AR3/antigenic domain B and AR4) revealed that the vast majority of antibodies (73%) targeted the AR3/antigenic domain B region. Additionally, antibodies targeting AR4 were present in 3 out of 4 individuals while no antibodies were found detecting epitope I/antigenic domain E or II/antigenic domain D only. Other studies will be necessary to confirm these observations and determine whether broadly neutralizing antibodies to other regions are involved in spontaneous clearance. The predominance of AR3- and AR4-directed B-cell responses in HCV clearers was confirmed using antibodies derived from monoclonal B-cell culture. Out of 12 isolated antibodies, including all antibodies directed against AR3, 10 showed broad neutralization of GTs 1a, 1b, 3a and 4a. Furthermore, 8 out of 12 antibodies neutralized at least 1 neutralization-resistant HCVpp with 2 of these, 1 AR3- and 1 AR4-specific antibody neutralizing all analyzed strains. In 1 of the clearers, AR3-specific memory B-cells were still present 5.2 years post clearance of primary infection suggesting long-lasting immunity against HCV infection. However, it should be noted that not all AR3/antigenic domain B antibodies have broad neutralization profiles.[13]Keck Z.Y. Saha A. Xia J. Wang Y. Lau P. Krey T. et al.Mapping a region of hepatitis C virus E2 that is responsible for escape from neutralizing antibodies and a core CD81-binding region that does not tolerate neutralization escape mutations.J VIrol. 2011; 85: 10451-10463Google Scholar What is the clinical impact of these findings? By directly comparing the B-cell responses of individuals clearing 1 or multiple HCV infection(s) with those of individuals who develop chronic infection, Merat and Bru et al. confirm that the early development of broadly neutralizing antibodies can confer protective immunity against HCV and thus reinforces the concept that the development of a protective vaccine is feasible. Furthermore, by identifying the broadly neutralizing epitopes associated with viral clearance they provide highly valuable information for structure guided vaccine design: Antigens containing the antigenic regions AR3/antigenic domain B and AR4 could serve as a starting point for the development of novel vaccine candidates.[14]Fuerst T.R. Pierce B.G. Keck Z.Y. Foung S.K.H. Designing a B cell-based vaccine against a highly variable hepatitis C virus.Front Microbiol. 2017; 8: 2692Google Scholar This is supported by previous findings that antibodies targeting AR3 and AR4 act synergistically15Giang E. Dorner M. Prentoe J.C. Dreux M. Evans M.J. Bukh J. et al.Human broadly neutralizing antibodies to the envelope glycoprotein complex of hepatitis C virus.PNAS. 2012; 109: 6205-6210Google Scholar, 16Carlsen T.H. Pedersen J. Prentoe J.C. Giang E. Keck Z.Y. Mikkelsen L.S. et al.Breadth of neutralization and synergy of clinically relevant human monoclonal antibodies against HCV genotypes 1a, 1b, 2a, 2b, 2c, and 3a.Hepatology. 2014; 60: 1551-1562Google Scholar and that some antibodies directed against AR3/antigenic domain B have been shown to present a high barrier against escape mutations.13Keck Z.Y. Saha A. Xia J. Wang Y. Lau P. Krey T. et al.Mapping a region of hepatitis C virus E2 that is responsible for escape from neutralizing antibodies and a core CD81-binding region that does not tolerate neutralization escape mutations.J VIrol. 2011; 85: 10451-10463Google Scholar, 17Velazquez-Moctezuma R. Galli A. Law M. Bukh J. Prentoe J. Hepatitis C virus escape studies of human antibody AR3A reveal a high barrier to resistance and novel insights on viral antibody evasion mechanisms.J Virol. 2019; 93Google Scholar The experience with other HCV B-cell vaccine candidates has shown that it is possible to elicit neutralizing B-cell responses with HCV E1/E2 based approaches. However, many of these responses have shown to be isolate-specific.[18]Ray R. Meyer K. Banerjee A. Basu A. Coates S. Abrignani S. et al.Characterization of antibodies induced by vaccination with hepatitis C virus envelope glycoproteins.J Infect Dis. 2010; 202: 862-866Google Scholar On the plus side, structure guided vaccine development has shown to be a successful strategy to elicit broadly neutralizing antibodies for other pathogens such as HIV,[19]Jardine J. Julien J.P. Menis S. Ota T. Kalyuzhniy O. McGuire A. et al.Rational HIV immunogen design to target specific germline B cell receptors.Science. 2013; 340: 711-716Google Scholar influenza A virus and respiratory syncytial virus. It remains to be determined whether B-cell focused approaches are able to provide immunity to HCV infection or whether an efficacious vaccine will also have to induce CD4 and CD8 based T-cell responses, as indicated by some of the previous studies. Ultimately, the findings provided by Merat and Bru et al. support the concept that a B-cell vaccine against HCV infection is feasible and most likely will have to induce broadly neutralizing antibodies targeted against AR3/antigenic domain B and preferably also against AR4. The latter offers the advantage of very high conservation among different HCV genotypes[20]Cowton V.M. Singer J.B. Gifford R.J. Patel A.H. Predicting the effectiveness of hepatitis C virus neutralizing antibodies by bioinformatic analysis of conserved epitope eesidues using public sequence data.Front Immunol. 2018; 9: 1470Google Scholar and thus might provide a very broad protection profile. This work was supported in part by National Institutes of Health grants U19-AI123862 (TFB, SKHF) and R01-AI132213 (SKHF), by ARC Foundation for Cancer Research (THERAHCC to TFB), by the European Union ( ERC-AdG-2014-671231-HEPCIR and H2020-2015-667273-HEP-CAR to TFB) and by the German research foundation (DFG − 395783133 to FW). This work has been published under the framework of the LABEX ANR-10-LABX-0028_HEPSYS (TFB) and benefits from funding from the state managed by the French National Research Agency as part of the Investments for the Future Program. The authors declare no conflicts of interest that pertain to this work. Please refer to the accompanying ICMJE disclosure forms for further details. The following are the Supplementary data to this article: Download .pdf (.25 MB) Help with pdf files Supplementary Data 1 Cross-genotype AR3-specific neutralizing antibodies confer long-term protection in injecting drug users after HCV clearanceJournal of HepatologyVol. 71Issue 1PreviewHepatitis C virus (HCV) is one of the major global public health problems, with 71 million chronically infected people worldwide, which results in 350,000 to 500,000 liver-related deaths per year.1 Current direct-acting antiviral (DAA) treatment is very effective in clearing infection.2 However, despite high DAA efficacy, treatment alone is unlikely to eliminate HCV by the year 2030 as envisioned by the World Health Organization (WHO),1 since treated individuals may become reinfected if exposure continues. Full-Text PDF