Using a highly sensitive in vitro infection assay developed as described previously (3
), we found in this study that (i) human blood contains components that facilitate HCV infection; (ii) the measured neutralizing activity of serum is the result of a combination of two opposite effects, true neutralization and facilitation of infection; and (iii) the kinetics of serum neutralization inversely correlate with HCV replication kinetics in patients with acute HCV infection.
An important finding in this study is the demonstration of the capacity of human serum from infected and noninfected individuals to facilitate in vitro infection by HCV pseudoparticles generated with E1-E2 glycoproteins derived from different HCV genotype and strains, including some retrieved from our patients' sera. This suggests that a human serum component(s) may facilitate HCV infection in vivo, as is already known for several other viruses. Facilitation of HCVpp infection, however, appears to be related to the specific interaction of E1-E2 envelope glycoproteins with this component(s). Indeed, it was not observed when using pseudoparticles generated with glycoproteins derived from alternative enveloped viruses (e.g., influenza virus, vesiculovirus, and retrovirus). In addition, the degree of facilitation depended on the HCV genotype or strain (Fig. ), i.e., on the envelope glycoprotein sequence. In this respect, it is interesting that the infectivity of HCVpp displaying the E1-E2 glycoproteins from virus strain B, which persisted in patients, was more efficiently facilitated by noninfected sera than those displaying glycoproteins from virus strain A, which was controlled during the acute phase.
The nature of the blood component(s) involved in facilitation of HCV infection remains unknown. Virus-specific antibodies have been reported to enhance viral infectivity both in vitro and in vivo (25
). Indeed, viruses from various families elicit antibodies that enhance infectivity through the binding of virus-antibody complexes to cellular Fc receptors (expressed in, e.g., monocytes/macrophages) via the Fc portion of the antibodies (24
). Fixation of the C3 or C1q complement proteins, activated by virus-antibody complexes, can also facilitate virus entry, as shown for the antibody-dependent, complement-mediated enhancement of infection of human immunodeficiency virus (19
) and Ebola virus (54
). Finally, in vitro enhancement of human immunodeficiency virus infection via an antibody-independent mechanism that involves receptors of the classical and alternative complement pathways has been reported (5
). However, none of these previously described mechanisms appears to be involved in the facilitation of infection by HCV pseudoparticles observed in this study, because heat-treated—decomplemented—sera from noninfected donors displayed the same levels of facilitation and because facilitation was not observed when the HCVpp were incubated with normal purified human immunoglobulin or monoclonal antibodies. On the other hand, the existence of infection-facilitating serum components is reminiscent of the fact that HCV circulates in vivo as complexes including serum proteins. Separation of infected blood samples by ultracentrifugation in sucrose or cesium chloride gradients indeed revealed two fractions in which HCV is abundant (6
). The first, at a density of ~1.25, contains viral particles mainly linked to immunoglobulins that are poorly infectious. The second, at a density of <1.06, contains viral particles that are mostly lipoprotein associated and infectious. The amount of HCV RNA in these fractions is highly variable and depends on the disease stage (60
) and on the virus genotype (29
). Whether the association of HCVpp with such serum components may occur in vitro upon their incubation with human serum and subsequently facilitate infection warrants further investigation.
Although definitive confirmation that human serum may facilitate HCV infection will require a reliable cell culture system to amplify wild-type virus and recover plasma-free particles, our results strongly suggest that the capacity of HCV to interact with an infection-facilitating serum factor(s) represents an essential component of sustained HCV infection. This not only may contribute to the “masking” of the virions from the immune system (47
), but may also represent a novel pathway of infection that exploits soluble serum factors for cell entry. This notion is supported first by our observation that facilitation of infection correlated with sustained high replication levels in the patients from group 2. Our data also suggest that the rise in neutralizing responses during the course of infection is associated with the disappearance of serum-facilitating effects, as is clearly seen in patients from group 2, such as Pt-8, Pt-9, and Pt-10. Whether facilitation of infection is eliminated in these cases or is still there but is overwhelmed by a counteracting neutralizing force remains to be determined.
The second important finding in our study is the inverse relationship between HCV RNA levels and the level of neutralizing responses in patients' sera. This inverse relationship was observed in the two groups of patients, including patients who underwent profound HCV RNA fluctuations and ultimately evolved toward HCV RNA clearance from serum (group 1) and patients who maintained high replication levels throughout the entire follow-up period (group 2). Patients from group 1 indeed elicited a neutralizing response that inversely followed HCV RNA kinetics, whereas the patients from group 2 remained with undetectable neutralizing response and exhibited a facilitating effect of their sera on HCVpp infectivity. The reason why the latter group of patients failed to raise a neutralizing response is unclear. It is not related to their inability to induce a humoral immune response, since these patients seroconverted during the study period for the same antibodies and with the same amplitude as the patients from group 1. The difference could eventually be explained by the fact that a majority of the two groups of patients were infected by either of two distinct HCV strains. This might suggest that different viruses bear intrinsic properties influencing their abilities to raise a neutralizing response upon infection. In this respect, chimpanzee challenge experiments with the two HCV strains from this study would be of major interest.
The observation of an inverse relationship between HCV RNA and neutralizing response kinetics at the acute phase of infection in our hemodialysis patients should be interpreted cautiously in a context where the role of humoral responses in the control of HCV infection is largely unknown. While vigorous and broad T-cell responses are involved in viral clearance (40
), a role for antibodies in protection against natural HCV infection has been difficult to establish. Nevertheless, there is evidence that polyclonal antibodies to HCV can be protective. Immunoglobulin preparations manufactured before the screening of plasma donors for HCV became common practice were shown to protect recipients against HCV infection (17
). Indeed, broadly reactive neutralizing and protecting antibodies were found in experimental immune globulin preparations made from anti-HCV-positive donations, as well as in a commercial immune globulin product, Gammagard, prepared from unscreened plasma (62
). Subsequent products prepared from pooled plasmas from which anti-HCV-positive donations had been excluded were reported to transmit HCV to recipients, potentially as a result of the removal of neutralizing antibodies (62
). In addition, studies of chimpanzees have also shown that anti-HCV immunoglobulins or hyperimmune sera can delay or prevent infection when the virus is inoculated after or at the same time as the antibodies (16
). However, whether a neutralizing response could be elicited at the acute phase of natural HCV infection and the precise role of neutralizing antibodies in the transient or sustained control of viral replication during acute hepatitis C remain unclear. In experimentally inoculated chimpanzees, Logvinoff et al. (35
) found that none of three animals with acute resolving infection developed neutralizing antibodies and, for other animals, that such antibodies could be detected in sera of acutely infected animals who did not resolve infection. This suggested that neutralizing antibodies did not play a critical role in the resolution of acute HCV infection, at least in the chimpanzee model. In contrast, our study shows for the first time the early emergence of a neutralizing response in patients who apparently evolved toward a control of viral replication, with strong responses concomitant with steep HCV RNA decreases (>4 log units) together with ALT normalization. The correlation of a relatively strong neutralizing response with a substantial loss of viremia was corroborated by the observation that, in the second group of patients, failure to reduce HCV RNA levels was associated with a lack of detection of a neutralizing response in blood from these patients.
At the present time, however, it is not possible to establish whether HCV RNA drops were actually the unequivocal consequence of the increase in neutralizing responses. In other words, whether neutralizing responses played a major role in the control of viral replication in group 1 patients is unclear. The fact is, however, that only the patients who could mount an efficient neutralizing response were able to control viral replication, whereas those who had no detectable neutralizing response continued to display very high levels of viral replication. Neutralizing antibodies alone do not appear to be able to control viral replication and lead to definitive HCV clearance. Indeed, cases of spontaneous resolution have been reported in HCV-infected agammaglobulinemic children (1
), suggesting that control of HCV may occur independently of antibodies, at least in a limited number of patients. Furthermore, it has been shown that chimpanzees vaccinated with recombinant HCV glycoproteins that induced high-titer antibodies were partially protected against a subsequent low-dose homologous HCV challenge (7
). However, experimentally infected chimpanzees and naturally infected humans could be reinfected with homologous and heterologous HCV strains, suggesting that humoral immunity that develops after spontaneous resolution of acute hepatitis C is not sterilizing (15
). The role of escape mutation selection, particularly in the HVR1 region, in the establishment of chronic infection has been suggested, both at the acute phase of infection and in patients receiving alpha interferon-based antiviral therapy (13
), a hypothesis that will be tested in the present series of patients. At this stage, our preliminary observation of an inverse relationship between HCV RNA and neutralizing response kinetics in acutely infected patients is challenging, yet further characterization of neutralization responses during acute hepatitis C is crucial to understanding HCV pathogenesis and developing efficient vaccines.