EXPERIMENTAL IMMUNOBIOLOGY

P.D. Acott, P.A. O'regan, A.O. Skotnicki, J.F.S. Crocker
Pediatric Nephrology, IWK Health Center, Halifax/NS/CANADA

Body: Introduction: We have established two renal cell lines with chronic BK virus (BKV) infection to investigate the antiviral properties of immunosuppressive drugs. Previous work has demonstrated in vitro antiviral effects of cyclosporine A (CsA) and mycophenolate acetate in contrast to viral propagation effects of steroids. Methods: Vero cells (Green monkey kidney cell origin) and PT cells (human proximal tubular cell origin) were each maintained as a monolayer on 24-well planar plates and/or Transwells^TM with removal of supernatant (SN) weekly for 4 week chronic BKV cellular infection after initial 2 hr incubation with ~ 6.5x10⁵ copies of BKV with either VJ patient isolate [VJ] or NCCR rearranged highly replicative VJ isolate [VJ-NCCR]. The Roche Lightcycler 2.0 was used for real time PCR quantification of the BKV DNA content of the culture supernatant (SN) and of the cells. Cell quantity and viability was assessed using a Countess^TM automated cell counter. CsA and/or Tacrolimus (Tac) stability in SN was confirmed by tandem mass spectrometry. High level replication was defined as > 10⁴ BKV DNA copies/ml at Day (D) 28. Results: Mean BKV DNA levels in Vero cells were significantly (p<0.01) higher in SN (D1-D7, 14, 21, 28) and cells (D28) when infected with VJ-NCCR as compared to VJ isolate [VJ-NCCR: SN D1-D7= 1.00x10⁰-1.96x10⁵, SN D14=1.85x10⁷, SN D21=3.49x10⁷, SN D28=2.55x10⁹, Cell D28=7.36x10⁹ versus VJ: SN D1-D7=2x10⁰-8.27x10⁴, SN D14=5.76x10³, SN D21=2.27x10², SN D28=2.9x10¹, Cell D28=1.36x10³ ] . Vero cells infected with VJ on planar plates had high level BKV replication in 43 of 102 wells (42%) by D28 reduced significantly by CsA (200 - 3200 ng/ml) with high level replication in 8 of 65 (12%) of wells (X² = 16.68, p<0.001). Tac treated (4-256 ng/ml) VJ infected Vero cells on planar plates had no significant antiviral effect with 24 of 57 (45%) achieving high level replication by D28 (X² = 0.18, NS). PT cells on planar plates infected with VJ had significantly lower risk of high level BKV replication (1 of 19 = 11%) compared to Vero cells (X² = 10.01, p<0.01). Growth of Vero cells infected with VJ in a polar environment (Transwell^TM) compared to planar environment had dramatic reduction of high level replication (0 of 38 = 0%) by D28 (X² = 22.05, p<0.001). Conclusions: This is the first demonstration of differential calcineurin inhibitor effects on BKV replication in vitro, with no evidence of Tac anti-viral BKV effect, despite re-confirmation of CsA suppression of BKV replication. We demonstrated cell type influences replication with proximal tubule phenotype (PT) cells more effective in control of BKV replication than Vero cells. Cellular micro-environment also is very important to BKV replication as demonstrated by better containment of Vero cell associated escape to high level BKV replication, simply by growing cells in a polar environment in Transwells^TM. Further understanding of local renal cell injury and phenotype contributions to BKV replication dynamics along with awareness of immunosuppressant viral propogation and/or anti-viral effects will be important to a full understanding of mechanisms of BKV replication in the transplant population.

Disclosure: All authors have declared no conflicts of interest.