The role of neutralizing antibodies during acute and chronic viral infection remains an important question and has generated controversial results. Initially, the presence of neutralizing antibodies was shown to control the HCV load and to contribute to viral eradication in patients capable of clearing the infection . In other studies, the appearance of neutralizing antibodies was delayed and restricted to IgG1 antibodies in patients who develop a chronic infection [2, 21]. The chimpanzee model has been critical for the study of HCV transmission and host immune responses; however, neutralizing antibodies were not detected in some animals that resolved their infection – suggesting a minimal role in viral clearance, as also observed in human studies [14, 15]. Experimentally infected chimpanzees and naturally infected humans can be re-infected with homologous and heterologous HCV strains, suggesting that the humoral immunity that develops after spontaneous resolution of acute hepatitis C is not sterilizing [22–24]. During chronic infection in humans, the presence and/or production of neutralizing antibodies do not suffice for curing the infection but could regulate the spread of the virus. Thus, it can be postulated that during chronic infection, viral mutants can continuously escape the renewed production of neutralizing antibodies.
Retroviral pseudoparticles have been used to develop a very interesting tool for measuring neutralizing antibodies in vitro . The assay has demonstrated the presence of HCV-neutralizing antibodies in human sera with relatively high titers (>1:320) and broadly neutralizing activity against different HCV genotypes. However, this model does not represent genuine HCV virions; in particular, the budding of retroviral particles is thought to be very different and may involve a variety of cellular pathways. Characterization of infectious retroviral pseudotype particles bearing HCV glycoproteins have been shown to be very heterogeneous, and so it is possible that these pseudoparticles may not be as relevant as the native HCV virions .
The recent development of a cell culture model for HCV enables the production of native HCV virions that can be efficiently propagated in cell culture [17–19]. This cell culture system has allowed us to develop a neutralization assay for evaluating the level and the proportion of HCV-neutralizing antibodies in chronically infected HCV patients. We analysed a number of parameters (such as practicability, reproducibility and specificity) and tested the effect of a range of variables (viral inoculum size, incubation time, fixation and permeabilization methods, blocking and revelation reagents) on these parameters (data not shown). Overall, the neutralization assay described in this study performs similarly to standardized neutralization assays for many other viruses [26–28].
The assay relies on the ability of the specific JFH-1 genotype 2 viral strain to replicate and multiply on a Huh-7 human hepatoma cell line in a cell culture model, enabling the rapid detection of viral foci after 72 hours of infection. Moreover, no secondary foci were detectable at this time point. Fixation with paraformaldehyde and permeabilization with Triton X-100 were chosen in order to preserve antigenicity and prevent the cell monolayer from detaching during washes. Development with DAB peroxide substrate made it easy to count specifically coloured viral foci. The viral inoculum size is an important parameter; it has to be low enough to enable good assay sensitivity but high enough to produce a statistically significant number of foci, i.e. allowing the reduction in the number of foci (and thus the effect of neutralization) to be monitored. Thus, 100 FFUs were used as the inoculum in this neutralization assay.
In order to test different human samples, we had to take into account the ability of HDL to facilitate HCVcc entry via a mechanism which depends on expression of the scavenger receptor BI [11, 15, 16, 20]. Given HDL's role in HCV entry, immunoglobulins were purified from each serum sample prior to determination of the neutralizing antibodies titer; this frees the assay of the risk of non-specific neutralization activity of the serum via the effects of HDL, the complement system and/or serum amyloid A protein (SAA) .
The HCV neutralization assay exhibited good reproducibility, for both duplicate assays and independent tests. As expected, the intra-assay coefficient of variation (CV) was lower than the interassay CV. The test also showed good specificity, since there was no interaction with anti-HIV, anti-HBV or heterophile antibodies. Very low titers were found with HCV ELISA and RNA-negative samples, and the assay's cut-off was determined as the mean titer for negative samples plus two standard deviations (1.25 log, corresponding to a dilution of 1:18).
Given that only the JFH-1 strain of HCV genotype 2a was available for the assay, we evaluated the neutralization titer of sera from patients chronically infected with other HCV genotypes, i.e. 1, 2, 3, 4 and 5. Most of these sera were detected as positive by the neutralization assay, except for two sera from HCV genotype 1-infected patients. These two samples presented a high specific antibody ratio according to the ELISA but only very low inhibition by neutralization assay (far below the cut-off, in fact). We conclude that either the samples lacked neutralizing antibodies or that any such antibodies that were present did not cross-neutralize with HCV genotype 2a. The sensitivity was 100% – not only for genotype 2 (the genotype of the strain used for the assay) but also for other HCV genotypes (except genotype 1). HCV genotype 5 antibodies were also measured but there were too few samples for accurate testing. Moreover, the positive sera (96.5%) had comparable and significantly high titers (1.99 ± 0.63), whatever the genotype. This finding suggests that most neutralizing antibodies are cross-reactive. Another possibility is that most of the patients had been previously infected by a genotype 2 strain. However, this is unlikely because few genotype 2 strains are circulating in France . As expected for a neutralization test, the assay presented in the present study appeared to be very specific (independently of the genotype) and usable in most circumstances. For most viral infections, neutralization assays such as that described in this study are used as reference assays. Thus, we are confident that as other HCVcc genotypes become available, these assays will replace the pseudoparticle assay in the near future because they are probably more relevant. Our assay is somewhat time-consuming and could be simplified by using one dilution to count the foci; however, this type of "short cut" would make it difficult to extrapolate to the dilution neutralizing 50% of the inoculum. Another approach would consist in using recombinant HCV capable of expressing reporter genes (such as luciferase) in order to use a single dilution and obtain a quantitative result . However, further neutralization studies using other genotypes are needed in order to complete our observations and to characterize the homologous and heterologous potencies of polyclonal and monoclonal neutralizing antibodies.