Aurintricarboxylic acid increases yield of HSV-1 vectors

Pechan, Peter; Ardinger, Jeffery; Ketavarapu, Jyothi; Rubin, Hillard; Wadsworth, Samuel C; Scaria, Abraham

ATA effect on HSV-1 yield in V27 cells To test whether ATA could increase the yield of rHSV-1 d27-1 (d27-1) vector in V27 cells, ATA was applied to the media during the infection (ATA@step1) or dilution steps (ATA@step2) (Figure 1a ). Interestingly, ATA treatment delayed HSV-1 plaque formation or cell lysis in V27 cell monolayers. Cytopathic effect at the time of harvest, 72 hours postinfection was between 20 and 60% as compared with 100% cytopathic effect in the absence of ATA (data not shown). To determine which concentrations and conditions for ATA addition have impact on HSV DNase resistant particles per milliliter (DRP/ml) and plaque-forming units per milliliter (PFU/ml) titers, ATA was added at varying concentrations (0-60 µmol/l ATA) either in six-well plates in the first step (ATA@step1) (Figure 1b ) or in T150 flasks in both steps (ATA@step1 and ATA@step2) ( Figure 1c ). Both protocols show HSV yield increase, and the optimal conditions would be either with 50 µmol/l ATA during the infection step (50 µmol/l ATA@step1) that is further diluted to the final concentration of 20 µmol/l ATA or by adding ATA at dilution step (20 µmol/l ATA@step2) to achieve the final concentration of 20 µmol/l ATA (Figure 1b,c) . In this example, the supernatant titers were 2.7 ± 0.1 × 10 8 DRP/ml and 8.1 ± 0.7 × 10 7 PFU/ml in 50 µmol/l ATA@ step1 protocol, 2.0 ± 0.7 × 10 8 DRP/ml and 7.4 ± 1.4 × 10 7 PFU/ml in 20 µmol/l ATA@step2 protocol, as compared with 7.3 ± 0.6 × 10 7 DRP/ml and 2.9 ± 0.6 × 10 7 PFU/ml of untreated control (0 µmol/l ATA) (Figure 1c ). Results are expressed as means ± SD from two independent experiments (n = 2). Importance of serum presence in ATA-HSV protocol We tested the effect of serum-free media on DRP/ml and PFU/ml HSV-1 titers in the above-described ATA-HSV protocol, which used 10% fetal bovine serum (FBS) (Figure 2 ). In this example of ATA@ step2 protocol, serum-free media resulted in d27-1 titer reduction from 3.2 ± 0.1 × 10 8 DRP/ml and 5.4 ± 0.3 × 10 7 PFU/ml (10% FBS) to 1.1 ± 0.2 × 10 7 DRP/ml (0% FBS), where PFU/ml titer value was below the detection limit ( Figure 2 ). Even more dramatic effect of serumfree media was seen in the ATA@step1 protocol when both DRP/ml and PFU/ml titer values were below the detection limit (data not shown).