Calcium Spirulan from Arthrospira platensis (Spirulina) Prevents Herpes simplex Virus Type I Cell Entry in vitro

Objective Calcium spirulan (CaSP) has been shown to prevent host cell penetration by several enveloped viruses, including Herpes simplex virus type 1 (HSV-1), in in vitro experiments. We investigated how CaSP inhibits HSV-1 penetration into human keratinocyte cells by studying the interactions among CaSP, HSV-1, and keratinocytes in vitro . Methods After chemical characterization of the CaSP, we conducted two separate experiments. First, we added different HSV-1 viral loads to HaCaT cells that had been incubated with CaSP for 3 h. Second, different viral loads were incubated with CaSP for 3 h and then the resulting mixtures were added to the cells. The incubation period lasted 48 h. The HSV-1 median tissue culture infectious dose (TCID 50 ) was determined by measuring the cytopathic effect (CPE) index and using a colorimetric immunohistochemical (IHC) staining. CaSP degradation and penetration into keratinocytes were determined using an HLPC method developed in house. Results CaSP was detected in the supernatant but not in the cells. After pre-incubating the cells with CaSP, a viral load of 4.56 TCID 50 was inhibited by concentrations of ≥50 µg/mL of CaSP after 48 h of incubation (CPE ≤2). An activity against the highest HSV-1 TCID 50 (114) was observed only with 800 µg/mL of CaSP. Similar effects were observed after 48 h pre-incubation of HSV-1 with CaSP when ≥50 µg/mL of CaSP was added to a viral suspension of 4.56 TCID 50 . The higher viral load (TCID 50 114) was not inhibited. Sustained inhibition of all HSV-1 titers used (from 0 to 1000 TCID 50 ) was observed only at a CaSP concentration of 800 µg/mL, independent from the pre-incubation method. Conclusion CaSP does not enter keratinocytes and seems to interact with both cells and virus as viral inhibition was independent from the preincubation method.