Hematopoietic cancer cell lines can support replication of Sabin poliovirus type 1

Abstract

Viral vaccines can be produced in adherent or in suspension cells. The objective of this work was to screen human suspension cell lines for the capacity to support viral replication. As the first step, it was investigated whether poliovirus can replicate in such cell lines. Sabin poliovirus type 1 was serially passaged on five human cell lines, HL60, K562, KG1, THP-1, and U937. Sabin type 1 was capable of efficiently replicating in three cell lines (K562, KG1, and U937), yielding high viral titers after replication. Expression of CD155, the poliovirus receptor, did not explain susceptibility to replication, since all cell lines expressed CD155. Furthermore, we showed that passaged virus replicated more efficiently than parental virus in KG1 cells, yielding higher virus titers in the supernatant early after infection. Infection of cell lines at an MOI of 0.01 resulted in high viral titers in the supernatant at day 4. Infection of K562 with passaged Sabin type 1 in a bioreactor system yielded high viral titers in the supernatant. Altogether, these data suggest that K562, KG1, and U937 cell lines are useful for propagation of poliovirus.

Figure 1

Viral titer in CCID50 value (a) or D-antigen level (c) of Sabin poliovirus type 1 per mL during serial passaging of Sabin poliovirus type 1 in hematopoietic tumor cell lines. Vero cells were used as a positive control cell line, whereas CHO cells served as a negative control. For the first infection, cells were infected with an MOI of 1, and cells together with supernatant were harvested after 3–6 days. After freeze-thawing of the material, half of it was used to reinfect fresh cells. This was repeated for 4 times. The virus titer in the 5 viral passages was determined by titration assay and the results are shown in (a). In (b) light microscopic images of uninfected or cells infected with Sabin poliovirus type 1 from passage 4 at day 6 (KG1 and K562) or day 3 (U937) are shown, whereas in (c) the D-antigen levels/10⁷ CCID50 for the fifth viral passage is shown.

Figure 2

Surface expression of CD155, the receptor for poliovirus, on Vero, CHO, and hematopoietic tumor cell lines using FACS analysis. In (a) CD155 expression from a representative experiment is shown. In the right upper corner the mean fluorescence intensity levels are shown. In (b) the mean percentage of CD155 expressing cells ± SD from 3–6 independent experiments is shown.

Figure 3

Replication kinetics of Sabin poliovirus type 1 (open circles) and adapted Sabin poliovirus type 1 that was serially passaged on Vero, K562, KG1, and U937 cells (black squares). Cells were infected with Sabin poliovirus type 1 from passage 0 or passage 5 and the titer in the supernatant was determined at days 1, 2, 4, and 7 after infection by endpoint dilution. The total amount of plaque forming units in these samples was determined and the mean ± SD from 3 independent experiments is shown.

Figure 4

Infection of Vero, K562, KG1, or U937 at an MOI of 0.01 with passaged Sabin poliovirus type 1 on the various cell lines results in a high virus titer in the supernatant after 4 or 7 days. After infection, both cells (black) and supernatants (grey) were harvested at day 4 and day 7, and the virus titer was determined in these samples. The total amount of plaque forming units in these samples was determined and the mean ± SD from 3 independent experiments is shown.

Figure 5

Efficient replication of Sabin poliovirus type 1 in K562 cells grown in Cultibags. K562 cells were infected with an MOI of 0.01 of passaged Sabin poliovirus type 1 when cells reached a concentration of 1.2 × 10⁶ cells/mL and samples of the supernatant were taken daily. In (a) and (b) the CCID50/mL and the D-antigen/mL at the various time points are shown, respectively. The mean ± SD from 3 independent experiments is shown.

Figure 6

Surface expression of CD54, CAR, and CD81, the receptors for rhinovirus, coxsackie, adenovirus, and hepatitis C virus, on the hematopoietic tumor cell lines. The mean percentage of expressing cells ± SD from 3–6 independent experiments is shown.