Fafi-Kremer S, Fofana I, Soulier E, Carolla P, Meuleman P, Leroux-Roels G, et al. Viral entry and escape from antibody-mediated neutralization influence hepatitis C virus reinfection in liver transplantation. J Exp Med 2010;207:2019-2031. (Reprinted with permission.)
End-stage liver disease caused by chronic hepatitis C virus (HCV) infection is a leading cause for liver transplantation (LT). Due to viral evasion from host immune responses and the absence of preventive antiviral strategies, reinfection of the graft is universal. The mechanisms by which the virus evades host immunity to reinfect the liver graft are unknown. In a longitudinal analysis of six HCV-infected patients undergoing LT, we demonstrate that HCV variants reinfecting the liver graft were characterized by efficient entry and poor neutralization by antibodies present in pretransplant serum compared with variants not detected after transplantation. Monoclonal antibodies directed against HCV envelope glycoproteins or a cellular entry factor efficiently cross-neutralized infection of human hepatocytes by patient-derived viral isolates that were resistant to autologous host-neutralizing responses. These findings provide significant insights into the molecular mechanisms of viral evasion during HCV reinfection and suggest that viral entry is a viable target for prevention of HCV reinfection of the liver graft.
Recurrent hepatitis C after orthotopic liver transplantation (OLT) for hepatitis C–associated end-stage liver disease or hepatocellular carcinoma is a vexing clinical problem. Rapid reinfection of the liver graft by hepatitis C virus (HCV) particles in the blood is nearly universal, and the ensuing disease often runs an accelerated course quickly progressing to graft cirrhosis and retransplantation or death.1 In contrast to hepatitis B, no passive immunoprophylaxis is currently available. The first potent, directly acting antiviral drugs are expected to be licensed only in 2011, and they have not been evaluated in individuals with end-stage liver disease or in the peritransplant setting.
The difficulties in creating efficient immunoprophylaxis are in large part due to the high genetic variability of HCV: this relatively small enveloped virus with an approximately 10-kb nonsegmented, plus-strand RNA genome has a highly error-prone RNA-dependent RNA polymerase.2 As a result, HCV exists in the infected host not as a homogeneous population but rather as a large number of variants that are collectively called the quasispecies swarm.3 Therefore, HCV can rapidly respond to selective pressures to which it is subjected by antiviral drugs or cellular and humoral immune responses; this is conceivably a prerequisite for establishing and maintaining a chronic infection.4
For HCV to infect target cells, the viral envelope glycoproteins, E1 and E2, must interact with at least four (co)receptors on the hepatocyte surface: scavenger receptor BI, the tetraspanin CD81, and two components of the hepatic tight junction (claudin 1 and occludin).5 It has been shown that the humoral immune response is a major driver of HCV E1E2 evolution during chronic infection because antibodies capable of neutralizing most viral variants in the swarm are continuously generated, but neutralization-resistant minor variants present in the swarm then become dominant and allow the virus to escape neutralization.6 Nonetheless, broadly neutralizing antibodies against E2 can offer protection against HCV infection, at least in an animal model.7
The laboratory of Thomas Baumert (Institut National de la Santé et de la Recherche Médicale and University of Strasbourg, Strasbourg, France) isolated 439 HCV glycoprotein E1E2 clones from pre-OLT and post-OLT sera of six patients undergoing liver transplantation for HCV genotype 1b–induced cirrhosis.8 Phylogenetic analysis showed that in most patients (four of six), the number and diversity of viral variants present in the serum abruptly decreased directly after transplantation (Fig. 1 in Fafi-Kremer et al.8). Conversely, the composition of the viral population remained largely stable when time points early (7 days) and late (1 month and later) after transplantation were compared.
Such a decrease in quasispecies diversity after OLT had previously been observed; it is thought that OLT presents a genetic bottleneck through which only a limited number of selected variants can pass, with many others being eliminated.9-11 However, the mechanisms determining which variants are selected have never been addressed experimentally. Here the present study breaks new ground.
In an intriguing experiment, an immunodeficient mouse [urokinase plasminogen activator/severe combined immunodeficient (uPA-SCID)] into which human hepatocytes had been transplanted was inoculated with pretransplant serum from one of the patients, and the authors observed that the same viral variant became dominant both in the human patient after transplantation and in the serum and liver tissue of the de novo infected mouse (Fig. 1). This underscores that selection is based on characteristics of the viral isolate rather than chance. To determine which functional properties are selected as the quasispecies swarm passes through the genetic bottleneck of graft reinfection, the authors used patient-derived HCV E1E2 clones to generate HCV pseudoparticles (HCVpp).
HCVpp are lentiviral particles that carry HCV E1E2 instead of the human immunodeficiency virus glycoproteins in their envelope.12-14 They are produced when envelope-deficient lentiviral particles are allowed to bud from 293T cells overexpressing HCV E1E2. The target cell entry of HCVpp is then mediated purely by HCV E1E2. Moreover, HCVpp harbor a minimal lentiviral genome into which a reporter gene such as green fluorescent protein or luciferase has been inserted; this allows the easy detection of successful target cell entry. HCVpp have been proved to be a faithful model of the early steps in the HCV replication cycle and allow rapid testing of large numbers of different HCV glycoproteins.
While the authors were testing pretransplant and posttransplant E1E2 clones in the HCVpp system, they observed that the selected variants were different from the unselected ones in two respects: (1) the selected variants generated more highly infectious HCVpp (this suggested that they were more efficient in mediating viral cell entry), and (2) they were resistant to neutralization by antibodies present in the autologous pretransplant serum. Moreover, the authors devoted some effort to defining which regions of HCV E1E2 conferred these selected traits, and it appears that mutations in multiple regions throughout the glycoproteins, including but not limited to hypervariable regions 1 and 2 and known CD81-binding motifs, were causative.
Finally, to probe the feasibility of passive immunoprophylaxis as a strategy for preventing graft reinfection, the authors tested the ability of a broadly neutralizing monoclonal antibody against HCV E2 (AP33) and a monoclonal antibody against HCV coreceptor CD81 to block the entry of selected variants, and they found that both approaches efficiently neutralized the selected variants from all six patients.
This elegant study by Fafi-Kremer and colleagues8 is insightful with respect to both clinical hepatology and basic infection biology.
From a clinical point of view, these data indicate that passive immunoprophylaxis in the transplant setting may be a feasible approach to preventing graft reinfection. By evolving to exploit gaps in the neutralizing antibody response by the infected host, a limited number of HCV variants successfully establish an infection in the new liver, but they are clearly susceptible to neutralization by broadly reactive anti-E2, such as the monoclonal antibody AP33 used in this study. This is in line with earlier data showing that escape mutants from chronically infected individuals are usually efficiently neutralized by heterologous serum from other chronically infected individuals.6, 15 Unfortunately, initial attempts at passive immunoprophylaxis of OLT recipients with either pooled anti-HCV–positive serum or monoclonal antibodies against E2 have been largely unsuccessful.16, 17 However, in recent years, more broadly and potently neutralizing antibodies against E1 or E2 that block diverse HCV genotypes have been developed.18 For one of these, efficacy in an in vivo model of HCV infection has been demonstrated.7 Similarly encouraging in vivo data from a mouse model have been reported for HCV blocking antibodies against CD81.19 Genetic variability is thought to be less of a concern when antibodies against a host encoded target are being used, although this potential advantage may be offset by a greater risk of side effects.
From the perspective of basic infection biology, this study offers a unique peek at the events surrounding the transmission of a highly variable viral pathogen. In vivo, HCV replicates primarily in hepatocytes, and whether viral replication also occurs in extrahepatic sites such as the lymphoid or central nervous system is unclear. Thus, we could still speculate that the selection of HCV variants follows very similar criteria during graft reinfection and during the transmission of HCV to a new host; in both cases, a naive liver is infected by virus particles from the bloodstream. The major difference is that in the case of reinfection, the infectious dose is very large, and the host's immune system has already been challenged by HCV. It is not very surprising that resistance to neutralizing antibodies present at the time of OLT and the ability to efficiently enter cells should be competitive advantages for viral variants in both situations, but an experimental demonstration of this had thus far been lacking. Even in the context of other viral infections, selection criteria during transmission events have rarely been defined rigorously. The most comprehensive information comes from the human immunodeficiency virus field: there it is well established that variants using chemokine (C-C motif) receptor 5 and not chemokine (C-X-C motif) receptor 4 as a coreceptor are almost always the ones that are transmitted and that predominate early during infection.20 Escape from neutralizing antibodies also seems to confer a selective advantage.21 To what extent efficient receptor usage plays a role is controversial.20
The study under discussion is not without limitations. First, the HCVpp system does not allow the assessment of genetic variations potentially conferring a selective advantage outside E1 and E2. Second, although the data hint at the feasibility of passive immunoprophylaxis for preventing graft reinfection, further proof-of-concept studies in animal models or HCV-infected patients are needed. Nonetheless, the authors offer a very fine example of reverse-translational or bedside-to-bench research, in that they have taken advantage of a unique opportunity presented by clinical medicine to gain insights into basic biological mechanisms. We hope that these insights will in turn inform clinical developments and the design of therapeutic strategies for preventing recurrent hepatitis C. Clearly, the long-term goal is to block reinfection and use OLT as an opportunity to cure both the long-term sequelae of chronic hepatitis and the underlying viral disease itself; this has already been achieved in the case of hepatitis B.