Human CD81 Knockout Cell Line-HEK293

The HCV pseudoparticle (HCVpp) system is a widely used platform to study cellular entry, screen for novel entry inhibitors, evaluate the phenotypes of E1 and E2 glycoproteins observed clinically, and most relevantly characterize the breadth of neutralizing antibodies induced in patients following vaccination and natural infection. Despite this, some patient-derived clones produce pseudoparticles that are either non-infectious or too low in infectivity for meaningful phenotypic analysis. Here, we show that endogenous expression of CD81, an HCV receptor and cognate binding partner for E2, in producer HEK 293T cells is deleterious to the infectivity of HCVpp recovered from most strains. Clones generated in CD81 knockout HEK293T cells are antigenically very similar to those generated from their matched parental cells and appear to follow the recognized HCV entry pathway. Deletion of CD81 does not significantly increase recovered titers of soluble E2 (sE2). The researchers also found that sE2 produced by Freestyle 293-F (293-F) cells contained mostly complex glycans, whereas sE2 produced by 293T displayed a heterogeneous mixture of complex glycans and high-mannose or mixed glycans. Furthermore, sE2 produced in 293T cells was superior in antigenicity; exhibiting increased binding to conformational antibodies and the large extracellular loop of CD81. In summary, this work describes an optimal cell line for the production of HCVpp and reveals that the antigenicity of sE2 produced in 293T and 293-F cells is not identical.

Here, researchers observed that HCVpp harvested from both CD81 knockout 293T lines had higher infection rates across the panel compared to matched viruses prepared in parental cells (Figure 2b). Improved infection was significantly more pronounced with HCVpp produced in CD81 knockout HEK293T cells compared to equivalents prepared in parental cells (Figure 1c). Finally, researchers prepared a panel of HCVpp expressing E1E2 in CD81 knockout HEK293T (293T^CD81KO) cells observed in HCV+ individuals in the Australian Prison Hepatitis C Incidence and Transmission Study (HITS-p) cohort. Upon infection readout, an increase in the signal-to-noise ratio (S/N) was observed for the majority of screened clones prepared in 293T^CD81KO cells. Surprisingly, 5 of the 22 screened clones that were previously identified as non-infectious were now infectious (S/N ≥5), and 8 of the additional 22 screened clones had at least a 5-fold increase in S/N. Finally, a comparison of the calculated S/N of all screened clones in all three panels showed an increasing trend in infectivity after HCVpp production in 293T^CD81KO cells (Figure 1d). Taken together, the 293T^CD81KO cells produced here can be a useful resource for HCV research.

Figure 1. HCVpp made in CD81 knock-out 293T cells exhibit enhanced infectivity. (Kalemera M D, et al., 2021)