3. Results and Discussion
Five cell lines (CHO, Vero, HEK-293, HT-1080 and MRC-5) were inoculated in duplicate with SARS-CoV-2 at two different MOI values of 1 and 0.01, respectively, and incubated for 14 days. During the incubation period the cells were periodically observed for CPE, as an indicator for viral infection in the same way as performed during routine AAT.
No CPE was detected throughout the incubation period for CHO cells, HEK-293 cells, HT-1080 cells and MRC-5 cells. In contrast, Vero cells displayed a CPE already on day 2 (MOI=1) or day 3 (MOI=0.01) post infection (p.i.). The permissiveness of Vero E6 and CCL81 cells for SARS-CoV-2 infection and production of CPE has already been demonstrated (Harcourt et al., 2020), and thus Vero CCL81 cells were used in this study as positive control, as well as for quantitation of SARS-CoV-2 replication in the other cell lines evaluated: the presence of infectious virus in the cell culture supernatant was determined at regular intervals throughout the incubation period by TCID50 assay, starting with the inoculum preparations and up to the last day of incubation (day 14).
For Vero cells inoculated with MOIs of 1 and 0.01, this analysis showed increasing SARS-CoV-2 titers in cell supernatants from day 2 and day 3 p.i., which peaked on days 6 and 7 p.i., at mean titers of 6.1 and 5.7 log10 TCID50/mL, and by 14 days p.i. slightly dropped by on average of 0.7 and 0.8 log10, respectively (Table 1).
For the CHO, HT-1080 and MRC-5 cell lines, no SARS-CoV-2 infectivity was detected by titration on Vero cells throughout the entire incubation period, which indicates that SARS-CoV-2 cannot replicate in these cells.
For HEK-293 cells inoculated at MOI=0.01, SARS-CoV-2 infectivity was never detectable in culture supernatants. After inoculation at MOI=1, however, low and constantly declining SARS-CoV-2 infectivity titers were detected until day 7 p.i. (Table 1). Whether these declining levels of virus reflect residual inoculum which may be more effectively bound by the HEK-293 as opposed to the CHO, HT-1080 and MRC-5 cell layers, or even some limited yet abortive replication as observed for other HEK-293-derived cells (Harcourt et al., 2020) remains to be determined.
In addition to the monitoring for the development of any CPE, the cells and supernatants were assayed at the end of the 14 days incubation period in the two standard AAT readouts, i.e., HAD to the cells, and HA activity in the supernatant, using human and guinea pig erythrocytes. This panel of altogether three assay readouts (CPE, HAD, HA) reflects exactly what is routinely performed during quality control testing of biomanufacturing fermenter harvests by using at least three different indicator cell lines (Figure 1). The choice of detection cells lines for AAT includes per FDA guidance (i) the type of cells used for biopharmaceutical production, (ii) a human diploid cell line, e.g. MRC-5 cells, and (iii) a monkey kidney cell line, e.g. Vero cells (US Food and Drug Administration, 1993).
For SARS-CoV-2 inoculated CHO cells, HT-1080 cells, HEK-293 cells and MRC-5 cells, virus was not detectable by any of the three AAT readouts, i.e. CPE as monitored throughout the two weeks of incubation and HAD / HA at the end of the incubation period. For Vero cells though, SARS-CoV-2 replication was, in addition to detection by CPE, detected by HAD with human erythrocytes (MOI=1 only). These findings confirm that AAT would confidently detect a SARS-CoV-2 contamination in a biomanufacturing cell culture tested, so long as Vero cells are included as a detector cell line.
Entry of SARS-CoV-2 into susceptible cells is facilitated by the same receptor as for SARS-CoV, i.e. the Angiotensin-converting enzyme-2 (ACE 2) (Hoffmann et al., 2020; Zhou et al., 2020), which suggests that the spectrum of susceptible cells is similar for SARS-CoV and SARS-CoV-2. High abundance of ACE2 as a cellular surface protein on Vero cells (Kaye, 2006) does correlate well with their susceptibility to infection with SARS-CoV-2. In contrast, the expression of ACE2 at low levels on CHO cells (Warner et al., 2005) might be the reason that SARS-CoV-2 replication in these cells was not observed. HEK-293 cells carry ACE2 at higher levels (Warner et al., 2005), which may facilitate binding and possibly entry of SARS-CoV-2 into HEK-293, followed by minimal and ultimately abortive replication (Table 1), or modest replication in HEK-293T cells (Harcourt et al., 2020).